Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRm160), an SR protein with a PWI RNA-binding motif, causes defective development only in male flies and results in abnormal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, we first identified the genome-wide RNA-binding characteristics of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. We then validated this binding through both in vitro gel-shift assay and in vivo splicing of minigenes and found that SRm160 level affects AS of many transcripts. Furthermore, we identified 492 differential binding sites (DBS) of SRm160 varying between the two sex-specific cell lines. Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160’s RNA-binding specificity and regulation of AS in Drosophila sex determination and development.