Chronic liver injury induces liver fibrosis that must be diagnosed and treated within an appropriate timeframe. Otherwise, it can easily evolve into liver cirrhosis, or other more severe liver diseases. In recent years, fluorescence imaging and spectral analysis techniques have greatly promoted the detection and research of liver fibrosis diseases because they offer rapid, simple, and non-instrusive detection, and thus have great potential. This review highlights the latest achievements of fluorescence imaging and spectral analysis techniques in the investigation of tissue, cell, and molecular mechanisms of liver fibrosis. The research difficulties and potential application prospects of the technique regarding liver fibrosis are also discussed.