In order to explore the in vitro incubation technique of fertilized eggs of Portunus trituberculatus, this study firstly screened the most suitable isolation solution for fertilized egg blocks, then conducted in vitro incubation studies of fertilized eggs at different developmental stages and transmission electron microscopy observation of fertilized egg membranes after treatment with the isolation solution, and finally measured the larval viability of fertilized eggs treated with the isolation solution after hatching. The results showed that papain was an ideal separation solution, and the separation rate was more than 99% with a concentration of 0.09 g/mL and separation time at 30 min. The highest hatching rate of 89.0%±3.3% was recorded for in vitro embryos in zoea stage, compared to 70.0%±4.8% for the control group. The lowest hatching rate for in vitro embryos was observed at the cleavage stage, which was 58.0%±3.9%, and that for the control group was 31.0%±2.3%. In general, compared with the control group, the hatching rate of the treatment group increased significantly; the observation results of transmission electron microscopy also supported this phenomenon with the structure of fertilized egg membrane being loose and its thickness decreased after the separation solution treatment; exposure to air, formalin solution stress and behavioral test were used to evaluate the quality of larvae in different treatment groups. The results showed that there was no significant difference in the vitality of larvae in treatment group and control group. In conclusion, the isolation solution obtained in this study can effectively improve the hatching rate of fertilized P. trituberculatus eggs without affecting the larval vitality, and our results can provide a reference for the in vitro embryos hatching of P. trituberculatus and other crustaceans.