Brackishwater catfish, Mystus gulio, is a commercially important fish of Southeast Asia. Farming of this fish is hindered due to unavailability of hatchery-produced seeds. In this study, three different experiments were conducted to determine suitable inducing hormone, operational sex ratio (OSR), and oocyte diameter for induced breeding of M gulio. In experiment one, human chorionic gonadotropin, HCG (5, 10, 15, 20 and 25 IU g−1), luteinizing hormone-releasing hormone analogue, LHRHa (2.5, 5, 7.5, 10 and 15 μg g−1) and commercial hormone, CH (a combination of LHRHa and domperidone) (10, 20, 25, 30 and 40 μl g−1) were tested separately. In experiment two, male and female-biased sex ratios, such as 1 M: 1F, 1 M: 2F, 1 M: 3F, 3 M: 1F, 2 M: 1F were used to ascertain correct OSR. Experiment three was conducted with four different size groups of oocytes (750–800, 801–850, 851–900, and 901–950 μm), and administration of primary and secondary doses of HCG (10 IU g−1 body weight) at 24 h interval. All the experimental treatments were conducted in triplicates (n = 3). Half the hormone dose of female was given to male in all the experiments. Result revealed that the effective doses of HCG, LHRHa and CH were 10 IU g−1, 5 μg g−1 and 20 μl g−1, respectively. Further, male biased sex ratio of 2 M: 1F caused significantly (p < 0.05) higher relative fecundity (169 eggs spawned g−1 fish), fertilization rate (77.33%), hatching rate (71.00%) and larval survival (31.67%). Female with minimum oocyte diameter of 851 to 950 μm, characterized by the presence of peripheral nucleus, is ideal for induced spawning. Oocyte with a further smaller diameter size of 801–850 μm required both primary and secondary doses of hormone injection. It is more economical to use CH for mass production of healthy larvae.