Sensitive metabolite detection in biofluids is crucial in in-vitro detection and commonly relies on the designed materials-based platform. However, metabolic analysis in biofluids is hindered by laborious sample pretreatment due to its complexity and sample abundance. Herein, we construct a mesoporous platinum (Pt) nanospheres system to enhance the laser desorption/ionization process for sensitive metabolites analysis. We successfully synthesize porous Pt nanoparticles with tunable surface structure by a simple surfactant-direction method. The nanoparticles promote the laser desorption/ionization process, demonstrating its sensitivity to standard metabolites (with a limit of detection down to 1.74 pmol, 100 times higher than traditional organic matrices). Notably, our platform directly realizes the sensitive detection of metabolites in biofluids (serum, plasma, urine, and cerebrospinal fluid) without sample pretreatment due to the size-selective effect of the porous structure. Our work will contribute to advancing techniques for metabolic analysis and developing novel materials-based platforms for in-vitro diagnostics.