In this study, a 3D SiO2 (M−SiO2) monolith was modified with four noble metals, namely, Au, Ag, Pt, and Pd nanoparticles (NPs), and was explored as a surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) substrate for forensic detection of drugs and pesticides. The loading of the NPs, reducing agent concentration, and reaction time were optimized using UV–vis spectroscopy. The prepared M−SiO2 monoliths were extensively characterized using different analytical techniques. The surface area of the pristine SiO2 monolith increased slightly upon introduction of the NPs, while the morphology of the SiO2 surface did not change after deposition. The effect of the NP loading and deposition method on SALDI-MS analysis of drugs and pesticides was investigated, and the results proved that layer-by-layer deposition provided the best SALDI performance. Among the prepared M−SiO2 monoliths, Au-SiO2 exhibited superior SALDI performance in terms of the signal-to-noise (S/N) ratio. Furthermore, the effect of NaCl salt was scrutinized using Au-SiO2 monolith, which enabled sensitive detection of the drugs and pesticides even in the presence of high salt concentrations. Finally, Au-SiO2 substrate was applied to the analyses of non-biological and biological samples. Specifically, drug-spiked carbonated malt drink and pesticide-spiked blood serum were analyzed using minute amounts of sample. The prepared substrate offered sensitive detection of the analytes with a limit of detection (LOD) of 100–1000 ng/mL and 1–100 ng/mL with a relative standard deviation (RSD) of 7.21–22.22% and 2.68–26.29% for the drugs and pesticides, respectively, indicating good spot-to-spot repeatability. Thus, the proposed method can be used as an efficient approach for forensic analysis of trace evidence owing to its simplicity and minimum sample volume.