This study investigated the effectiveness of Artificial Intelligence-Based Virtual Laboratories on Senior Secondary School students’ Biology practical skills in Abakaliki Education Zone, Ebonyi State, Nigeria. Two research questions and three hypotheses guided the study. The study adopted a quasi-experimental research design, specifically the non-equivalent control group pre-test post-test design. A sample of 175 Senior Secondary II students was selected through purposive sampling. Abakaliki Local Government Area was sampled out of the four Local Government Areas in Abakaliki Education Zone, and two public secondary schools were selected. Intact classes were used, with 96 students assigned to the experimental group and 79 students to the control group. The Biology Practical Skills Inventory (BPSI), a 20-item performance-based instrument rated on a 5-point scale with a maximum score of 100, was used for data collection. The instrument was validated by three experts in Biology Education and Measurement and Evaluation, and its reliability was established using Cronbach’s Alpha, yielding a coefficient of 0.87. The experimental group was taught using Artificial IntelligenceBased Virtual Laboratories, while the control group received instruction using the Conventional Laboratory Method. Research questions were answered using mean and standard deviation, while Analysis of Covariance (ANCOVA) was used to test the null hypotheses at 0.05 level of significance. The findings revealed that students exposed to Artificial Intelligence-Based Virtual Laboratories significantly outperformed those taught using the conventional method in Biology practical skills. The study also found no significant gender difference and no significant interaction effect between teaching method and gender. Based on the findings, the study recommended that Biology teachers, school administrators, and educational policymakers should integrate AI-based virtual laboratory tools into secondary school instruction to enhance students’ practical competence in Biology.