Abstract: As a widely utilized bactericidal and disinfectant agent, malachite green (MG) presents significant risks to ecological systems and public health due to its environmental persistence, bioaccumulation potential, and well-documented carcinogenic properties. The compound has been frequently detected in aquatic environments and agricultural products, thereby raising substantial concerns regarding its implications for food safety and ecosystem stability. Conventional mass spectrometry instruments often exhibit insufficient spatial resolution, which limits their capacity for detailed molecular imaging at the single-cell level—a critical capability for advancing toxicological research and elucidating cellular responses to environmental stressors. To overcome these analytical constraints, this study developed a novel ion source configuration that incorporated a precision tapered fiber integrated with an optimized projection optical system. This advanced design not only enhances laser energy concentration but also serves as an effective physical barrier, effectively preventing neutral molecular species and particulate contaminants from entering the ionization region. As a result, the system demonstrates improved signal stability, measurement reproducibility, and long-term operational reliability. The newly developed ion source was integrated into a customized time of flight mass spectrometer (TOF MS), leading to the creation of an advanced imaging platform termed the tapered fiber projection laser desorption time of flight mass spectrometry (TFPLD-TOF MS) system. The platform was employed to analyze HeLa cells exposed to varying concentrations of MG. Morphological assessments revealed distinct concentration-dependent apoptotic features, including cellular shrinkage, membrane blebbing, and nuclear fragmentation—all of which are clear indicators of MG-induced cytotoxic effects at the cellular level. Moreover, by utilizing gold nanoparticles (Au NPs) as a high-performance nano-matrix to enhance laser desorption efficiency, the system achieved high resolution mass spectrometry imaging (MSI) capable of simultaneously mapping the spatial distribution of the carcinogenic drug and a diverse array of endogenous lipid metabolites. This approach enables the identification and spatial localization of 28 distinct lipid species across multiple lipid classes, including triglycerides (TGs), phosphatidylinositols (PIs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), and ceramides (Cers). These findings provide valuable insights into MG induced lipidomic alterations, metabolic pathway disruptions, and subcellular compartment specific responses to toxic exposure. The TFPLD-TOF MS platform consistently demonstrates high analytical sensitivity, robust anti-contamination properties, and exceptional spatial resolution throughout the experimental evaluations. These characteristics make it as a powerful and reliable analytical tool for advanced toxicological studies, particularly in elucidating molecular mechanisms underlying single-cell responses to environmental contaminants and bioactive compounds. The platform holds significant potential for broader applications in cellular biology, clinical diagnostics, pharmaceutical development, and environmental health monitoring, offering new opportunities for advancements in molecular toxicology and precision medicine.