Label-free visualizing the prediabetic microenvironment of adipose tissues provides a less invasive alternative for the characterization of insulin resistance (IR) and inflammatory pathology. Here, we successfully identified the differentiable features of prediabetic adipose tissues by employing the metabolic imaging of three endogenous fluorophores NAD(P)H, FAD, and lipofuscin-like pigments. We discovered that 1040-nm excited lipofuscin-like autofluorescence could mark the location of macrophages. This unique feature helps separate the metabolic fluorescence signals of macrophages from those of adipocytes. In prediabetes fat tissues with IR, we found only adipocytes exhibited a metabolic fluorescence profile different from that of normal adipocytes. When mice have inflamed fat tissues, both adipocytes and macrophages possess this kind of metabolic change. Based on spatial fluorescence metabolomics, we developed an innovative approach to diagnose prediabetes, providing insights into diabetes prevention strategies.