Spinibarbus hollandi is an important commercial aquaculture species in southeastern China, but with long maturity period and low egg laying amount. However, there has been little study of its gonad development and reproductive regulation, which limits aquaculture production. Here, for the first time, gonadal transcriptomes of male and female S. hollandi were analyzed. A total of 167,152 unigenes were assembled, with only 48,275 annotated successfully. After comparison, a total of 21,903 differentially expressed genes were identified between male and female gonads, of which 16,395 were upregulated and 5508 were downregulated in the testis. In addition, a large number of differentially expressed genes participating in reproduction, gonad formation and differentiation, and gametogenesis were screened out and the differential expression profiles of partial genes were further validated using quantitative real-time PCR. These results will provide basic information for further research on gonad differentiation and development in S. hollandi.