Human TMPRSS3 is a member of the TypeⅡ Transmembrane Serine Protease (TTSP). This was the first description of a serine protease involved in deafness. Previously, we have found many mutations in the TMPRSS3 genes from screening of the 230 children with non-syndromic deafness (14/230; 6.09％). In addition, we have investigated and confirmed the effect of mutations in TMPRSS3 in yeast and Xenopus. Therefore, identification of TMPRSS3 orthologies in different species raises interesting question. In this study, we examined the function of TMPRSS3 orthologies to human and mouse genome in zebrafish genome. First, we found tmprss4a and tmprss4b are similar to human TMPRSS3 and mouse Tmprss3 using approaches of bioinformatics. By RT-PCR, tmprss4a expresses from early to late stage and tmprss4b expresses before 9 hour post-fertilization. Here, we present tmprss4a and tmprss4b gene expression of embryo by whole mount in situ hybridization. The results show that zebrafish tmprss4a express ubiquitously but have the strong signal at otic vesicle at 14-16 hpf. At 24 hpf and 48 hpf, the expression of tmprss4a is not only in otic vesicle but also in lateral line with strong signal. In contrast, tmprss4b express ubiquitously at early development stages. On the contrary, there is no any significant signal at 10-22 somite and 24 hpf and 48 hpf. We also present morpholino knockdown studies targeting tmprss4a and tmprss4b. Embryos at one to two cells stage injected with tmprss4a ATG MO or tmprss4a e9i8 MO will have bending notochords, shortened anterior- posterior axes and heart edema at 72 hpf. With tmprss4b ATG MO or tmprss4b e7i6 MO, embryos show axes bending less than 90° or greater than 90° or heart edema at 72 hpf. However, function of tmprss4a and tmprss4b are direct towards hearing in zebrafish need to be investigated further. At the same time, we found a new zebrafissh gene, tmprss3, from the recent version of NCBI databases. Phylogenetic analysis of transmembrane serine protease protein family show tmprss3 was the most similar to human TMPRSS3. But the protein sequence of tmprsss3 was incomplete. Our results showed 160 nucleotides were detected by 5’ RACE technology. But the sequences we found do not have any start codon for translation. Further investigation for looking for a translational start site was required.