The low level of scientific literacy and the suboptimal integration of 21st-century skills in Biology learning highlight the need for innovative and interactive digital learning media. This study aimed to develop an interactive learning-based e-module integrated with 21st-century skills (4C: Critical Thinking, Creativity, Communication, and Collaboration), evaluate its validity and practicality, and analyze its effectiveness in improving senior high school students' scientific literacy on ecosystem topics. The study was conducted from January to May 2026 using a Research and Development (R&D) approach with the ADDIE model (Analysis, Design, Development, Implementation, Evaluation). Product effectiveness was evaluated using a quasi-experimental design with a pretest-posttest control group design. The participants comprised 160 tenth-grade students from SMAN 1 Kasokandel and SMA Islam Al Mizan, Majalengka Regency, with 40 students in the experimental class and 40 students in the control class at each school. Data were collected through expert validation sheets, student response questionnaires, learning implementation observation sheets, and scientific literacy tests, and were analyzed using the Wilcoxon Signed Rank Test and the Mann-Whitney U Test. The results showed that the e-module achieved a very high validity level, with an average feasibility percentage of 92.9%, comprising 93.7% from material experts, 87.5% from media experts, and 97.7% from Biology learning experts. The e-module was also considered highly practical, as indicated by a 100% positive response across all questionnaire items. The effectiveness test demonstrated a significant improvement in scientific literacy among students in the experimental classes at both schools (Asymp. Sig. = 0.000 < 0.05). The experimental classes achieved N-Gain scores of 0.73, categorized as high, at SMA Islam Al Mizan and 0.68, categorized as moderate, at SMAN 1 Kasokandel, exceeding the control classes' scores of 0.33 and 0.19, respectively. Therefore, the interactive e-module integrated with 4C skills is valid, practical, and effective in supporting the improvement of senior high school students' scientific literacy on ecosystem topics. ABSTRAK Rendahnya literasi sains serta belum optimalnya integrasi keterampilan abad ke-21 dalam pembelajaran Biologi menuntut pengembangan media pembelajaran digital yang interaktif. Penelitian ini bertujuan untuk mengembangkan e-modul berbasis pembelajaran interaktif yang terintegrasi keterampilan abad ke-21 (4C: Critical Thinking, Creativity, Communication, dan Collaboration), menguji validitas dan kepraktisannya, serta menganalisis efektivitasnya dalam meningkatkan literasi sains siswa SMA pada materi ekosistem. Penelitian dilaksanakan pada Januari–Mei 2026 menggunakan metode Research and Development (R&D) dengan model ADDIE (Analysis, Design, Development, Implementation, Evaluation). Efektivitas produk diuji menggunakan quasi-experimental design dengan pola pretest-posttest control group design. Subjek penelitian melibatkan 160 siswa kelas X dari SMAN 1 Kasokandel dan SMA Islam Al Mizan Kabupaten Majalengka, masing-masing terdiri atas 40 siswa kelas eksperimen dan 40 siswa kelas kontrol. Data dikumpulkan melalui lembar validasi ahli, angket respons siswa, lembar observasi keterlaksanaan pembelajaran, dan tes literasi sains, kemudian dianalisis menggunakan Wilcoxon Signed Rank Test dan Mann-Whitney U Test. Hasil penelitian menunjukkan bahwa e-modul memiliki tingkat validitas sangat tinggi dengan rata-rata persentase kelayakan 92,9%, yang meliputi penilaian ahli materi sebesar 93,7%, ahli media sebesar 87,5%, dan ahli pembelajaran Biologi sebesar 97,7%. E-modul juga dinyatakan sangat praktis berdasarkan respons positif siswa sebesar 100% pada seluruh butir angket. Hasil uji efektivitas menunjukkan adanya peningkatan literasi sains yang signifikan pada kelas eksperimen di kedua sekolah (Asymp. Sig. = 0,000 < 0,05). Nilai N-Gain kelas eksperimen mencapai 0,73 dengan kategori tinggi di SMA Islam Al Mizan dan 0,68 dengan kategori sedang di SMAN 1 Kasokandel, lebih tinggi dibandingkan kelas kontrol yang masing-masing memperoleh 0,33 dan 0,19. Dengan demikian, e-modul interaktif terintegrasi keterampilan 4C dinyatakan valid, praktis, dan efektif untuk mendukung peningkatan literasi sains siswa SMA pada materi ekosistem.