Abstract: To improve the efficiency of the CRISPR/Cas9 gene editing system, we designed and optimized Cas9 protein, and purified HeiCas9 protein with high activity was obtained by E. coli codon optimization, prokaryotic expression, immobilized metal affinity chromatography, molecular sieve chromatography, and ultrafiltration centrifugation concentration. In this study, HeiCas9 protein, Cas9 protein (T brand), and zcas9 mRNA were combined with the gRNA of tyr, the key gene for melanin synthesis in zebrafish, to knockout tyr gene in zebrafish embryos at different concentration gradients, and the tyr gene editing efficiency was compared in different groups. At a working concentration of 400 ng/µL, HeiCas9 demonstrated significantly higher gene editing efficiency than T brand Cas9 protein while maintaining lower embryotoxicity. We further used HeiCas9 protein to knockout the genes dnd of Gobiocypris rarus and prkdc of Carassius auratus, with promising results. In summary, we have successfully generated highly active HeiCas9 protein, and validated its utility for efficient gene editing in multiple small fish embryos models under laboratory conditions.