Abstract: According to the amplification character of polymerase in polymerase chain reaction, the PCR circulating parameters were studied and the extension temperature gradient was eliminated during amplification. The two-temperature gradient PCR method was used not only in both single PCR and multiplex PCR amplification study, but also in the PCR amplification for bovine samples of genome, fibroblast, embryos respectively. At last, the stable, simple, fast method of two-temperature gradient PCR for sexing bovine pre-implantation embryos was obtained. At the same time, the amplification ability of two-temperature gradient multiplex PCR was detected, as a result, it indicated that less than 633bp chain could been synthesized successfully in the two-temperature gradient PCR condition.