Biological Science at the H.S level is the initial stage for career development in medicine and other domains of Bioscience. However, students frequently encounter multifaceted barriers that delay learning. This cross-sectional study investigated the barriers perceived by H.S students in learning Biological Science, with a focus on institutional types, instructional language, regional, and gender-based differences. A sample size of 106 Class XII science stream students under the state educational board Assam Higher Secondary Education Council (A.H.S.E.C) from Kamrup Metropolitan and Kamrup Rural,Assam.. A 30-items scale the Barriers in Learning Biological Science Scale (BLBSS) was developed, validated, and administered. The data were collected from four different schools (two public, two private),the data collected was not included in the range of normality, therefore a non-parametric test was found to be statistically useful for the inquiry. Mann-Whitney U test was used to examine difference among the variables. The findings revealed that students from public institutions stated a higher learning barriers compared to their private school counterparts (z(106)=-6.556, p=.000). A significant disparity was seen among students from English medium and Assamese institutional language ( z (106)= -3.401, p= 0.001). A substantial regional discrepancy was observed, with students from Kamrup Rural experiencing barriers in learning Biological Science than Kamrup Metropolitan (z (106)=-5.271, p=0.000). The finding revealed that no gender difference was found (z(106) = -.751, p = 0.452). The findings underscore the urgent need for prior interventions in public and rural schools; meanwhile introduce English as an institutional language to teach biological science at higher secondary level to enhance biology learning outcomes.