Objective To simulate the detection of plankton-aerosols generated by the rearing and flushing of rhesus monkeys using standard operating procedure ( SOP ) for microbes in an animal biosafety level 3 ( ABSL-3) laboratory. Methods The ground excreta and filter basket at the outlets of the monkey cages were measured during the stationary or feeding state. The SOP for feeding the monkeys and cleaning the cages was either not strictly followed or was strictly followed. A bacillus bacterium was used as the indicator microorganism and aerosols were collected using the Sedolis Airport MD 8 portable plankton sampler. The detection method included live cell cultures and PCR detection. Results (1) In the stationary state, there were < 5 bacteria. (2) In the feeding state, the bacterial culture count from the floor of the monkey cage was approximately 30 / dish. The yeast growth number was 1 / dish, and the bacterial count in the filter basket at the drain outlet was > 300 / dish accompanied by the growth of > 10 / dish. ( 3) The comparative test result demonstrated that the growth of bacteria and yeast was significantly reduced when strict SOP was followed for cleaning compared with not following the SOP. No bacterial DNA was detected on the absorbent mat. Conclusions The risk of microbiological contamination can be effectively controlled by strict implementation of SOPs for feeding and cleaning cages of rhesus monkeys in the ABSL-3 laboratory.