In this research, we developed and validate an instrument for assessing high-school students’ perceptions of socio-scientific issues (SSI)-based learning in biology. The development of the instrument was carried out using the following four steps: item formulation, content validation, construct validation and reliability calculation. According to an extensive literature review, 28 initial items were formulated for four SSI-based instruction aspects: (1) contextualisation of SSI, (2) student involvement, (3) student attitudes towards SSI-learning and (4) SSI-based learning objectives. Based on the pilot study conducted for construct validation, 24 items were successfully constructed for the four scales and subsequently administered in biology classrooms to 151 students. Factor analysis showed that each of the 24 items had a factor loading at least 0.40 on its own scale and no other scale. Moreover, the four scales accounted for 58.32% of the total variance and the alpha reliability coefficients for the scales ranged from 0.75 to 0.85. According to these findings, the SSI-based Learning Environment Inventory in Biology is valid, reliable and useful.