In order to explore the physiological response and tolerance mechanism of Bacillus subtilis after exposure to formaldehyde, laser tweezers Raman spectroscopy (LTRS) is employed to perform the experimental process in which germinated spores (g. spores) are stressed with different concentrations of formaldehyde for 2 h. The results show that both vegetative cells and g. spores have tolerance to formaldehyde, but the growth and physiological effect of g. spores stressed by different concentrations of formaldehyde are dissimilar. A lag phase of characteristic spectrum involved with various biomacromolecules is observed while g. spores are exposed to 0 mmol/L of formaldehyde medium from 0 to 0.5 h, subsequently, the Raman spectra peaks tend to rapidly ascend from 0.5 to 2.0 h. Characteristics and trend of Raman spectra variation are all from ascending to descending while g. spores are exposed to 0.4, 0.8, and 1.0 mmol/L of formaldehyde medium respectively for 2 h, and the corresponding inflexion of Raman spectra causes various components in vivo to decline where the time points are 1.5, 1.0, and 0.5 h, respectively. The analytic results of Raman spectra display that various components in vivo decrease gradually after 1.5 h while g. spores are stressed by 0.4 mmol/L of formaldehyde, implying that g. spores are harmed mildly. While g. spores are in 0.8 mmol/L formaldehyde medium, the band of nucleic acid decreases slowly at the very start, indicating that DNA cannot duplicate in the whole process. The bands of membrane phospholipids and C-S (protein) stretching mode descend obviously after formaldehyde stress for 0.5 h, which suggested formaldehyde could break down the membrane lipid hydrocarbon chains. While g. spores are in 1.0 mmol/L of formaldehyde medium, the high formaldehyde concentration makes a severe stress effect on cells, the content of biological macromolecules drops significantly after 0.5 h leading to cells deterioration gradually.