Tumor necrosis factor α(TNF-α) is an important inflammatory cytokine implicated in the pathogenesis of rheumatoid arthritis, septic shock and other diseases. In order to investigate the functional role of host TNF-α in Mycobacterium marinum (M. marinum) infection, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) technology was utilized to mutate the TNF-α gene in wild type (WT) zebrafish. A zebrafish containing a frame-shift mutation was successfully constructed, isolated, and named as TNF-α^－/－. It was found that the expression of TNF-α mRNA was significantly reduced in TNF-α^－/－ zebrafish by in situ hybridization (ISH). The proliferation of M. marinum in TNF-α^－/－ zebrafish was significantly elevated compared to WT zebrafish. The construction of TNF-α^－/－ zebrafish will allow further in-depth study on the function of TNF-α in mycobacterial infection and related immune regulation.