In this paper , the orthogonal experiment of three factors and three levels L9 ( 3 4 ) was adopted using blood corpuscle of Haliotis discus hannai which were in vitro immersing in PHA solution . There were three factors and three levels which included PHA treatment t ime : 6 h , 1 2h , 18 h ; PHA concentration : 0. 02%, 0. 04%, 0. 06% ; sampling time: 16: 00- 18: 00, 22: 00- 24: 00, 4: 00- 6: 00, respectively . The test s were repeated two times. The results showed that the optimal combination of three factors and three levels in the detection of triploid Haliotis discus hannai by using chromosome specimens from blood cells of living bodies was: PHA treatment time 18h, PHA concentration 0. 04% and sampling time 22: 00- 24: 00. The order of the three factors was PHA treatment time .. PHA concentration .. sampling time. Under the optimal combination of the three factors and three levels many clear, proper length and wel-l scattered metaphase chromosomes could be obtained. It is the best method of detecting the ploidy of living Haliotis discus hannai . Determining the relatively content of blood corpuscles.. DNA with flow cytometry could also make ploidy detection. But the device was expensive and the method was diff icult to be spread.