Laser tweezers Raman spectroscopy system is an optical technique combining the laser tweezers and Raman spectroscopy technique, which could be able to study recognition and detect constituent components of single biological cell in close to its natural state. Rat fetal liver stem cells at 10~21 days of gestation and adult rat hepatocytes were separated by collagenase digestion. Single fetal liver stem cell, hepatocyte and WBF344 rat liver stem cell line were detected by laser tweezers Raman spectroscopy technique, and their Raman spectrum was compared. It was indicated that the value of 1 336 cm－1 (assigned to Polynucleotide chain, DNApurine bases) and 1 446 cm －1(CH2 bending mode of proteins) band increased before 13th day and decreased after 14th day of gestation, and the mean of peak value of 1 336 cm－1 and 1 446 cm－1 bands at 13th day of gestation was the highest. The results showed that the Laser tweezers Raman spectroscopy system could differentiate fetal liver stem cells of different period of gestation from adult hepatocytes, and analyze differentiation and development mechanism of fetal liver stem cells, which lay a foundation for clinical application of hepatic stem cells therapy.