KARS encodes for both mitochondrial and cytoplasmic lysyl-tRNA synthetase, which is necessary for protein synthesis. KARS mutations have been reported to be involved in hearing loss, vision disorder, neuropathology, etc. However, there is no relevant animal experimental research in vivo so far. Here, we developed a zebrafish KARS null mutant line, offering a model for diseases due to KARS mutations. Zebrafish embryos with different genotypes (homozygous or wild type) were separately collected for each biological replicate at 5 days post fertilization (dpf). Proteomics of three biological replicates for the two groups was performed and analyzed. Comparisons between these two groups allow identification of enriched proteins and pathways in KARS mutants, which may underlie phenotypes of KARS deletion. Our dataset provides a valuable resource for understanding the pathology and molecular mechanisms of KARS deficiency.