【Objective】The objective of this study was to establish a rapid assay to detect complex vertebral malformation （CVM）gene carrier so as to gradually eliminate CVM gene carriers for a lower loss in the cattle-feeding industry.【Method】 In this study, a pair of primer and two TaqMan probes were designed according to the sequence of SLC35A3 gene from GenBank. A TaqMan real-time PCR assay for CVM gene was developed. DNA samples from clinical Holstein cattles were analysed by the assay, and the results were used for comparison with sequencing method.【Result and conclusion】 The assay could effectively differentiate mutant type, wild type and hybrid type of CVM gene. The sensitivity of three types was 1×10³，5×10³ and 1×10⁴ μL^-1, respectively.Six out of 292 tested Holstein cattles were identified to be CVM gene carriers. The results were consistent with the sequencing method.The TaqMan real-time PCR is a rapid and reliable method for extensive screening of CVM gene at the port of entry.