Laser tweezers Raman spectroscopy (LTRS) is used for real-time investigation of the overexpression of broad bean 14-3-3b soluble protein and inclusion body protein in the single living Escherichia coli cell at different temperatures after induction with isopropyl thiogalactoside (IPTG). The results show that 14-3-3b soluble protein and inclusion body protein have significantly different Raman characteristic peaks, which reflects that the two kinds of proteins have distinct conformations in their main chains and side chains. Intensity of the characteristic peaks of the inclusion body protein, including 900, 1033, 1328, 1415 and 1446 cm-1, gradually increases as the function of the culture time after IPTG is added at 28 ℃, however, intensity of the peaks of the soluble protein, including 763, 1002, 1363, 1451 and 1665 cm-1, increases in small increments relatively. Conversely, the growth rate of soluble protein peaks is higher than that of the inclusion body peaks obviously at 16 ℃, indicating that the recombinant protein is mainly formed as inclusion body at 28 ℃, and mainly as soluble protein at 16 ℃. Under lower temperature, the information of protein folding in precision can be reflected by the spectral changes and the correct protein folding into certain configuration is conducive to the formation of soluble protein in E. coli cells. In addition, the non-recombinant protein of the bacteria expression at 16 ℃ contains more cystine, which may be associated with protein folding. The LTRS is a useful tool for nondestructive, unmarked, efficient, real-time monitoring of overexpression processes of soluble protein and inclusion body protein in a single living E. coli cell.