Staphylococcal nuclease is a small globular protein, whose variants are widely used in the researches on protein folding. Different from methods and techniques reported in published papers, fluorescence dynamics of tryptophan residues in two staphylococcal nuclease (SNase) variants Δ+PHS and Δ+PHS+I92A were investigated by time-correlated single photon counting (TCSPC) and femtosecond fluorescence upconversion techniques, combined with UV absorption and steady-state fluorescence spectroscopy. Based on the analysis, structures and thermal stabilities of the two SNase variants were studied. The results proved that tryptophan could be used as an endogenous probe for the structural folding and thermal stability of the SNase variants. Decay associated spectra (DAS) of SNase variants showed different changing trends upon temperatures. According to this, structural folding and thermal stability of the two variants were analyzed. Time-resolved emission spectra (TRES) demonstrated the 05 ns continuous spectral relaxation process of tryptophan residue, in which the spectral shift showed the compactness difference of folding structures of the two SNase variants. In femtosecond upconversion results, DAS of 05 ps lifetime had “positive blue edge and negative red edge”, which showed relaxation effects on tryptophan residues in SNase variants. Moreover, the lifetime of 200 ps indicated the electron transfer between tryptophan residues and surrounding quenching group. Analysis of time-resolved anisotropy showed that the tryptophan residues had independent segmental motion in the protein system, and its intensity was related to the thermal stability of SNase variants and the overall effect of thermal motion. Time-resolved fluorescence measurement and analysis of tryptophan residue helped to investigate the relationship between structure and function of protein.