Encapsulation of Azo‐QM‐PN with DSPE‐PEG2000 results in the formation of the nanoprobe Azo‐QM‐PN NPs, which can effectively penetrate cell membranes, specifically illuminate tumor cells, monitor fluctuations in azo reductase levels, and deeply penetrate and image multicellular tumor spheroids, demonstrating potential for hypoxic tumor imaging. Here, NQO1 is linked to neoplasm.