Nitric oxide (NO) is a fundamental signaling molecule that regulates virtually every critical cellular function, and it is also a potent mediator of cellular damage in a wide range of conditions mainly via its secondary metabolite peroxynitrite (ONOO⁻). In this work, we present an o-phenylenediamine (OPD)-locked Si-rhodamine deoxylactam, i.e.deOxy-DALSiR, as a near-infrared fluorescent probe for the selective and sensitive detection of NO in living cells and bodies. Not only could the probe overcome the limitations suffered by widely used and commercialized OPD-type fluorescent NO probes, such as the possible interferences by dehydroascorbic acid/ascorbic acid/methylglyoxal (DHA/AA/MGO), pH-sensitive fluorescence output, and short excitation and emission wavelengths, but it can also avoid serious interference from cysteine (Cys) found in the rhodamine lactam-based fluorescent NO probes developed later. What’s more, the probe is fairly sensitive for NO, as evidenced by its rapid fluorescence response rate (within seconds), huge fluorescence off–on ratio (6300-fold), and ultra-low detection limit (0.12 nM). Its effectiveness and practicability have been demonstrated by the successful imaging of endogenous NO in RAW 264.7 macrophages, pancreatic β-cells, and endothelial EA.hy926 cells, as well as in inflamed and diabetic mouse models.