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The use of synthetic steroids of 17-Methytestosterone (MT) to produce a monosexual population of male Tilapia (Oreochromis niloticus) has been restricted because it can leave harmful residues in fish and the environment. One alternative that can be done is by using natural bioactive derived from plants with androgenic properties, such as Pasak Bumi (Eurycoma longifolia Jack). The aim of this research was to assay the use of methanol extract from Pasak bumi roots with immersion techniques for masculinization of tilapia. The research procedure was carried out with three treatment doses of methanol extract from the Pasak Bumi roots (namely 30, 60, and 90 mg/L), treatment dose amounted to 0 mg/L acted as a negative control and treatment of MT amounted to 50 mg/L acted as a positive control, with three replications on each treatment. Immersion was applied to larvae that was hatched out of egg yolk (seven day-old) for 24 hours and was maintained with a capacity of 50 larvae per aquarium during 60 days. The results showed that the treatment of the methanol extract of Pasak Bumi roots gave male sex ratios ranging from 72.66% - 76.33% higher than the negative control which showed the results of male sex ratios amounted to 49.19% and lower than the positive control of MT which the results amounted to 83.99%. The range of survival rate was from 80.67% - 85.33%, while the range of survival rate in the negative control was 83.33% and in positive control of MT was 82.67%. Thus, the methanol extract of Pasak Bumi roots can be used for masculinization of tilapia by immersion method with the best performance at a dose of 60 mg/L – 90 mg/L.