The bloodstain is one kind of the most important evidences in crime scenes. Laser-induced breakdown spectroscopy (LIBS) technology was used in this paper to distinguish bloodstains from analogs and determine the time since deposited (TSD) of bloodstains. Combined with support vector machine classifier, the identification results of bloodstains from analogs achieved 96% in 20 s. Four kinds of substrates were chosen to simulate the grounds and lethal tools in crime scenes. The linear relationship between single spectral line intensity and TSD was analyzed. The Pearson linear correlation coefficient (Pearson's r) calculated on the stone bricks calibration result is higher than 0.9. The spectral line intensity ratio was proposed to improve the linear calibration relationship with TSD. Building the calibration relationship between intensity ratios and TSDs, the Pearson's r and R 2 values for results on all four substrates were improved. For stone bricks, ceramic tiles, and copper plates, the correlation coefficients were all higher than 0.95. Especially for results on stone bricks, the Pearson's r achieved 0.99024 and the R 2 value achieved 0.97571. The optimal single lines for calibration on four substrates were all related to Fe element and the optimal line intensity ratios were all ratios of an Fe line to a line of other elements.