Tb3+‐doped zinc sulfide (ZnS:Tb3+) nanocrystals were synthesized by spray precipitation with sulfate‐reducing bacterial (SRB) culture at room temperature. The morphology of the SRB and ZnS:Tb3+ nanocrystals was examined by scanning electron microscopy, and the ZnS:Tb3+ nanocrystals were characterized by X‐ray diffractometry and photoluminescence (PL) spectroscopy. The PL mechanism of ZnS:Tb3+ nanocrystals was further analyzed, and the effects of Tb3+ ion concentration on the luminescence properties of ZnS:Tb3+ nanocrystals were studied. ZnS:Tb3+ nanocrystals showed a sphalerite phase, and the prepared ZnS:Tb3+ nanocrystals had high luminescence intensity under excitation at 369 nm. The main peak position of the absorption spectra positively blueshifted with increasing concentrations of Tb3+ dopant. Based on the strength of the peak of the excitation and emission spectra, we inferred that the optimum concentration of the Tb3+ dopant is 5 mol%. Four main emission peaks were obtained under excitation at 369 nm:489 nm (5D4→7F6), 545 nm (5D4→7F5), 594 nm (5D4→7F4), and 625 nm (5D4→7F3). Our findings suggest that nanocrystals have potential applications in photoelectronic devices and biomarkers.