As an organelle in cells, lysosomes play an important role in the degradation of biological macromolecules and pathogens. To elucidate the function of lysosomes in normal or disease states, recently, various fluorescent probes have been reported for imaging lysosomal analytes. However, because of the particularity of the lysosomal environment, most of the reported lysosomal fluorescent probes suffered from a series of practical issues such as easy diffusion, low detection signal-to-background ratio and false signal. To address these issues, based on an optimized in situ ordered assembly solid-state fluorophore HDPQ, we herein put forward a new strategy for the design of lysosomal enzymes probes. As a proof concept, we synthesized a fluorescent probe HDPQ-GLU for lysosomal enzyme β-glucuronidase (GLU). Experiment results displayed that compared with general lysosomal probe, the novel lysosomal probe not only exhibited excellent anti-pH interference ability and high signal-to-noise ratio in aqueous solution, but also has excellent long-term in situ imaging ability in the living system. Using this probe, we have realized high-fidelity and long-term in situ tracking GLU in lysosomes of living cells and evaluated the dynamic changes of GLU during the growth period of zebrafish. We anticipate that the new strategy based on the novel in situ ordered assembly solid-state fluorophore HDPQ may be a potential platform for developing fluorescent probes for high-fidelity imaging of lysosomal enzymes.