For the past several decades, most of the research studies on nanozymes have been aimed at improving their catalytic activity and diversity; however, developing nanozymes with strong catalytic activity and great specificity remains a challenge. Herein, a simple and efficient template synthesis method was used to synthesize bimetallic sulfide nanoparticles, NiCo 2 S 4 NPs, and prove that they have excellent peroxidase-like activity with good specificity. By regulating polyvinyl pyrrolidone (PVP) and hexadecyl trimethyl ammonium bromide as the templating agent, we have obtained the NiCo 2 S 4 (PVP) NPs with a high Ni/Co ratio, thus exhibiting superior peroxidase activity. In addition, the NiCo 2 S 4 NPs selectively catalyzed and oxidized colorless 3,3,5,5-tetramethylbenzidine (TMB). On being treated with H 2 O 2 , TMB turns blue while other substrates did not undergo the oxidation reaction under the same conditions, such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) and dopamine. The high specificity of NiCo 2 S 4 NPs is due to the strong electrostatic driving coordination between negatively charged NiCo 2 S 4 NPs and positively charged TMB. Due to the peroxidase activity of the developed NiCo 2 S 4 NPs, a simple, low-cost, and reliable colorimetric method was established. Simultaneously, this method for in situ quantitative monitoring of H 2 O 2 produced by MDA-MB-231 cells was also achieved. This study has provided a theoretical basis for the improvement of the activity and specificity of bimetallic sulfide nanozymes and may offer guidance for the further reasonable design of related materials.