Using an ultrasonication-assisted liquid exfoliation method, we have synthesized PtS2 nanosheets with good reproducibility. Herein, intrinsic peroxidase-like activity was for the first time demonstrated for PtS2 nanosheets, which can catalyze H2O2 oxidation of 3,3 ',5,5 '-tetramethylbenzidine (TMB) to generate a colored solution. The catalytic mechanism of PtS2 nanosheets was investigated, which indicated that acceleration of the electron transfer between TMB and H2O2 was the main reason for the peroxidase-like activity of PtS2 nanosheets. Based on these observations, we exploited PtS2 nanosheets integrated into dopamine-functionalized hyaluronic acid (HA-DA) hydrogel microspheres by droplet microfluidics to construct PtS2 nanosheet- and PtS2@HA-DA microsphere-based sensors for highly sensitive determination of H2O2. When coupled with glucose oxidase, we further developed two glucose sensors based on the above two methods. Among them, the linearity of the PtS2 nanosheet-based spectrophotometry was in the range of 0.5 to 150 mu M and the limit of detection as low as 0.20 mu M. The linearity of the microsphere-based colorimetry was in the range 200 to 12,000 mu M with a detection limit of 29.95 mu M. Both of the glucose sensors can be applied to the determination of glucose in human serum with reliable results and reproducibility.