Objective To investigate the positive and suspicious rates of BV, SRV, SIV and STLV-1 antibody of the cynomolgus monkeys introduced from Laos, dynamic monitoring of virus antibody during the establishment of SPF cynomolgus monkey population, and then comparing the positive rate of virus antibody in the baby monkeys between the common with SPF populations. Methods Continuous monitoring four kinds of virus in the monkeys was carried out using special reagent kits and analyzed the data. Results In the introduced cynomolgus monkeys (n=1998), the positive rate of BV antibody was 52.35%, suspicious rate of BV antibody was 8.31%, and negative rate of BV antibody was only 39.34%, the positive rates of SRV and STLV-1 antibody were 7.45% and 8.56%, respectively, and positive or suspicious SIV antibody in the cynomolgus monkeys was not detected. After screening and establishment of the SPF population, the positive rates of BV, SRV and STLV-1 antibody were 5.24%, 1.01% and 0.4%, respectively, in the year 2010. Then after five years of screening and elimination, by the end of 2014, the positive rates of BV, SRV and STLV-1 antibodies were 0.82%, 0.27% and 0.27%, respectively, and positive or suspicious SIV antibody in the cynomolgus monkeys was not detected. The BV antibody positive rate of the baby monkeys was 9.71% and the suspicious rate was 1.85% in the common population, but only 0.22% of the baby monkeys in the SPF population. Conclusions Continuous monitoring virus antibodies and eliminating positive and suspicious animals have important significance in the establishment of a SPF cynomolgus monkey population.