Despite therapeutic advances in glioma, glioma-related mortality rates remain high due to its extremely poor prognosis and high recurrence. Near-infrared (NIR) fluorescence imaging technologies, using the clinically approved fluorescent probe 5-ALA, have gained prominence in facilitating visualization of glioma resection. However, the false-positive and false-negative results of 5-ALA decrease its sensitivity and specificity in detecting glioma, thereby hampering its use in glioma surgical navigation. Herein, a novel molecular probe MPA-Pip-abt-510 labeled with a NIR fluorescent dye MPA was developed, and its ability to target the CD36 protein, which is upregulated in glioma, was assessed. Fluorescent-labeled probes, conjugated to the CD36-targeting ligand abt-510, demonstrated high specificity and selectivity for CD36-positive tumor cells in vitro and tumor tissue in vivo. Biodistribution analysis of MPA-Pip-abt-510 revealed high tumor-specific accumulation in tumors, accompanied by minimal nonspecific uptake in background tissues, yielding a signal-to-noise ratio (SNR) of 6.6 ± 0.4 in a U87 subcutaneous glioma model 10 h postinjection. Meanwhile, quantitative analysis validated the high uptake of MPA-Pip-abt-510 in the U87 orthotopic tumor model, with a tumor-to-brain SNR of 5.4 ± 0.5, enabling the accurate identification of tumor tissue for surgical navigation. Moreover, pathological analysis of tumor and healthy brain tissues unveiled well-defined tumor boundaries, highlighting the capacity of the MPA-Pip-abt-510 probe to precisely visualize the CD36 protein at the molecular level. Given its rapid tumor-targeting abilities, durable retention, and accurate outlining of tumor boundaries, MPA-Pip-abt-510 emerges as a promising CD36-targeted fluorescence contrast agent and expands the toolbox of glioma fluorescent probes for surgical navigation.