Increasing the bioavailability of immobilized phosphorus (P) in soil by phosphate-solubilizing bacteria (PSB) is an effective strategy for sustainable agronomic use of P and for mitigating the P crisis. Here, D₂O isotope labeling combined with single-cell Raman spectroscopy (Raman–D₂O) was developed as an efficient activity-based approach to characterizing the presence and activity of PSB in a culture-independent way. On the basis of the finding that PSB were significantly more active than non-PSB in the presence of insoluble P, a C–D Raman band from active assimilation of D₂O-derived D was established as a biomarker for both inorganic-phosphate-solubilizing bacteria and organic-phosphate-solubilizing bacteria. C–D ratios (intensities of C–D bands as percentages of the intensities of both the C–D and C–H bands) were further established as semiquantitative indicators of P-releasing activities because of the consistency between the C–D ratio and the concentration of solubilized phosphate or acid phosphatase activity as measured by conventional bulk assays. By applying Raman imaging, single-cell Raman–D₂O clearly discerned PSB in a mixed-soil bacterial culture and even in complex soil communities. Remarkable heterogeneity of microbial activity, ranging from 2 to 30% (close to that in medium without P and that in medium with sufficient soluble P, respectively), was revealed at the single-cell level and clearly illustrated the subpopulation of soil bacteria active in solubilizing P. This work not only enables probing PSB and their P-releasing activities but also opens a window to explore more diverse microbial resources when obtaining related isotope-labeled substrates is prohibitive.