The beef industry prefers male calves as they tend to have higher growth rates and feed efficiency for meat production. The dairy industry, however, prefers female calves which produce offspring and milk for dairy products. Thus, methods are required to determine the sex of spermatozoa in order to manipulate the sex of the offspring of livestock. The ability to sex spermatozoa has great potential for commercialization in the beef and diary cattle, thus a lot of the research has been conducted to develop and refine spermatozoa sexing technology. In this study, fluorescence in-situ hybridization (FISH) was performed on decondensed bovine spermatozoa using testis-specific protein, Y-encoded (TSPY) marker, as a screening method for the detection of Y-chromosome bearing bovine spermatozoa. The PCR-produced fragments of TSPY were cloned, transformed, cultured, and extracted according to a standard protocol. TSPY fragment-bearing plasmids were labelled with SpectrumRed-dUTP by nick translation labelling. The labelled probes were hybridized onto the pre-washed slides containing decondensed bovine spermatozoa for 72 hours. After post hybridization, the washed slides were counterstained using 4,6-diamino-2-phenylindole (DAPI II) and the FISH images were captured and analysed. The results showed only certain spermatozoa cells were hybridised with red signals, indicating the presence of Y-chromosome bearing spermatozoa. It is important to note that this chromosome-specific marker can be used to verify the sex of the flow cytometrically sorted spermatozoa and open the way of elucidation for TSPY to be used as a marker in sex determination.