Background/purpose. Higher-Order Thinking Skills (HOTS) development represents a critical challenge in biology higher education. While Project-Based Learning (PjBL) shows promise, empirical evidence remains fragmented across individual studies. This meta-analysis systematically synthesized evidence on PjBL effectiveness in developing HOTS among biology students and explored moderating factors influencing outcomes. Materials/methods. A comprehensive literature search across five databases following PRISMA guidelines yielded 2,847 records. Rigorous screening identified 42 high-quality experimental studies encompassing 5,247 undergraduate biology students across 18 countries. Meta-analytic procedures included standardized mean differences calculation, heterogeneity assessment, moderator analyses, and publication bias evaluation. Results. Meta-analytic synthesis revealed a large overall effect size (d = 0.847, 95% CI: 0.692-1.002, p < 0.001) favoring PjBL interventions over traditional instruction methods. Domain-specific analyses demonstrated consistently large effects across cognitive (d = 0.923), psychomotor (d = 0.862), and affective (d = 0.756) dimensions of HOTS development. Moderate heterogeneity (I[superscript 2] = 68.4%) reflected meaningful variation in implementation approaches while maintaining coherence sufficient for pooled estimation. Sensitivity analyses confirmed robustness of findings across different study characteristics, with no evidence of significant publication bias detected through funnel plot analysis and statistical testing. Conclusion. This meta-analysis provides compelling evidence that Project-Based Learning represents a highly effective pedagogical intervention for developing Higher-Order Thinking Skills in biology higher education contexts. The large, consistent, and robust effects observed across multiple domains and diverse implementation contexts support widespread adoption of well-designed PjBL interventions as a transformative strategy for preparing biology students to meet contemporary scientific challenges and collaborative problem-solving requirements.