Abstract: The optimal proliferation conditions of infectious spleen and kidney necrosis virus(ISKNV) in CPB cells with synchronous inoculation method were confirmed through detecting the viral copy numbers.The virus which proliferated with different FBS concentration,amount of inoculated virus,cell state,and viral harvest time was harvested and then DNA was extracted for qPCR detection.Results showed that all of the above factors had obvious influence on the ISKNV proliferation in vitro.It would obtain the highest amount of virus(2.54×10⁸ copies/mL) under the conditions that the MOI was 0.2,the virus was inoculated to the CPB cells cultured for 48 h at the middle-log phase and cultured with L-15 medium containing 6% fetal bovine serum and harvested after culturing at 28℃ for 9 days.The average cost of viral yield per yuan and per day was further analyzed.The results indicated that if the culture medium cost was only considered,it would obtain the highest viral yield(4.24×10¹¹ copies) per yuan as the above method;however,if it was as far as possible to shorten the time of cell culture and virus culture for saving time cost,it would obtain the highest viral yield per day(3.64×10⁷ copies) under the conditions that the MOI was 4,virus was inoculated to the CPB cells cultured for 24 h at the early-log phase and cultured with L-15 medium containing 10% fetal bovine serum and harvested after culturing at 28℃ for 4 days.In conclusion,this paper will provide the theoretical basis for ISKNV vaccine production with low cost through the comprehensive viral yield analysis of the medium cost and culture time cost.