Background: Femtosecond laser-induced breakdown spectroscopy (fs-LIBS) is a powerful tool for biomedical diagnostics because it provides real-time, in situ elemental analysis with high spatial resolution. This study used fs-LIBS to distinguish melanoma from normal dermis tissue. Methods: A crater size of approximately 20 μm and an enhanced signal-to-background ratio of critical emission lines were achieved by optimizing the key experimental parameters, such as laser energy, gate delay, and spectral normalization. Results: Principal component analysis and support vector machine algorithms were applied to the normalized spectra, resulting in 96.4% classification accuracy. Conclusion fs-LIBS, under the appropriate experimental conditions and data processing, provides precise tissue analysis and has significant potential for advancing bio-imaging and early cancer detection.