We demonstrate the application of multiphoton microscopy in diagnosing toxin- (CCl4-) induced liver fibrosis in mice. Although hepatocyte autofluorescence does not vary significantly, different degrees of necrosis and stellate cell proliferation at necrotic sites in livers with fibrosis (ex vivo) can be detected easily from multiphoton-induced autofluorescence images by use of 780-nm excitation. Our result suggests that multiphoton microscopy can be developed into an effective technique for the detection and diagnosis of liver fibrosis in vivo.