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Optimization and characterization of biodiesel production from fish oil extracted from rohu (Labeo rohita) processing waste were investigated in the present study. Wet reduction method showed highest oil yield (95.2%, v/v), compared to other methods (P < 0.05). Physicochemical properties of the oils were investigated before preparing biodiesel through transesterification reaction. Various alcohols, catalysts, reaction temperature and reaction time were evaluated for optimal biodiesel conversion of oil. The maximum yield of biodiesel (95.9%) was obtained with 0.75% KOH as catalyst, 1:0.5 (v/v) oil to methanol ratio and a reaction temperature of 55 °C for 60 min. TLC, FTIR and GC analysis suggested the triglycerides in the RPW oil have completely converted into biodiesel, which contained palmitic acid methyl ester and α-linolenic acid methyl ester as the dominant esters. Physicochemical characteristics of biodiesel …