The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement. However, common small-molecule intensity-based probes and most available genetically encoded F?rster resonance energy transfer (FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles. Therefore, we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72, referred to as ZnT72R, which has an approximate Kd that varies from 6.07 ± 0.28μmol/L to 7.84 ± 0.42μmol/L in vitro and a cytosol-calibrated Kd of approximately 55.56 ± 4.59μmol/L in HEK293 T cells. To pinpoint the free zinc concentration of insulin-storing vesicles, we initially targeted ZnT72R to beta-cell vesicles by fusing them to NPY (neuropeptide Y). Following NPY-ZnT72R labeling, the FRET intensity ratios of vesicles were quantified. We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice (28.30 ± 1.33μmol/L) was significantly lower than that of control mice (41.46 ± 3.53μmol/L).