Objective To investigate the optimal cryoanesthesia time for Aedes albopictus and Culex pipiens pallens and the survival status of them after cryoanesthesia, and to provide a scientific basis for anesthesia before mosquito bioassay. Methods A certain number of pupae of Ae. albopictus and Cx. pipiens pallens were put into different mosquito cages to obtain four cages each of emerged Ae. albopictus and Cx. pipiens pallens. At 2-3 days after emergence, they were stored in a freezer (-20±0.5)℃ for 5, 10, 15, and 30 s, respectively. They were taken out to observe the knock-down rate and first recovery time. And then they were given routine feeding in feeding rooms together with their respective control groups. Their survival status was observed and recorded. The median survival time and survival curve were compared between different freezing groups. Results The knock-down rates of Ae. albopictus after 5, 10, 15, and 30 s of freezing were all 100%. There was no significant difference in survival curve between the 5 s group and its control group (χ²=1.441, P=0.230), while the 10 s group and 15 s group had significantly shorter survival time compared with their respective control groups (χ²=24.407, P<0.01; χ²=38.375, P<0.01). There were no recovering mosquitoes in the 30 s group. Five cages of Ae. albopictus from the 5 s group were put on ice floe for 3, 5, 10, 30, and 60 min, respectively. The first recovery time and daily survival status were observed. The first recovery time of Ae. albopictus in the 3 min group was 145 s, and there was no significant difference in survival curve between the female mosquitoes in this group and those in its control group (χ²=2.463, P=0.117); however, the female mosquitoes in the 5, 10, 30, and 60 min groups had significantly lower survival curves compared with their respective control groups (χ²=15.141, P<0.01; χ²=22.518, P<0.01; χ²=30.242, P<0.01; χ²=43.672, P<0.01). The knock-down rates of Cx. pipiens pallens after 5, 10, 15, and 30 s of cryoanesthesia were 64.00%, 85.19%, 100%, and 100%, respectively. The first recovery times for the 15 s group and 30 s group were 3 s and 138 s, respectively. There was no significant difference in survival curve between the test groups and their control groups (χ²=7.225, P=0.124). Conclusion For Ae. albopictus, the optimal operation procedure for cryoanesthesia is 5 s freezing and 3 min placement on ice floe, while the optimal time for Cx. pipiens pallens is 30 s freezing.