Purpose: Conditional mutation is crucial for determining stage- and tissue-specific functions of genes. Herein, we developed and characterized an inducible knockout platform composed of the heat-inducible hsp70l promoter and clustered regularly interspaced shot palindromic repeats (CRISPR)/Cas9 system, an easy and efficient strategy for generating gene-modified cells and organisms. Materials and methods: We generated a heat shock-dependent Cas9 zebrafish embryo by inserting an hsp70l promoter-driven Cas9 transgene expression cassette through the Tol2 transposon system. To overcome the difficulty in identifying zebrafish germline integrations and facilitate observation of Cas9 expression, this cassette contains heart-specific cmlc2:EGFP and a selectable marker, P2A-mCherry. Real-time PCR and immunoblotting were performed to characterize heat-dependent, constitutive expression of Cas9 in zebrafish after heat shock. Furthermore, we designed and constructed plasmids containing zU6 promoter-driven tyr guide RNA and cmlc2:mCherry, which is a distinguishable marker used to confirm expression of gRNA. This plasmid was injected into one-cell fertilized Cas9 embryos. After heat shock, we estimated the efficiency of targeted mutagenesis of injected embryos through T7 endonuclease I assay and real-time PCRs. Results: Heat treatment of Cas9 transgenic embryos resulted in expression of mCherry fluorescence throughout the whole body without morphological abnormalities, and Cas9 protein was expressed 1 hr after the onset of heat shock, with high levels of Cas9 gene expression observed by 6-12 hr post-heat shock. In testing endogenous Tyr locus, we found that the mutagenesis efficiency reached approximately 50%. Mosaic phenotypes were observed in the injected embryos. Conclusions: The results indicate that our developed mutagenesis approach is efficient and could be used to generate conditional alleles throughout the zebrafish genome.