In the present study, we firstly developed a simple method using a conventional real-time PCR system to measure the serum autofluorescence and cfDNA-related fluorescence in PHC, liver cirrhosis (LC), chronic hepatitis (CH) patients and normal control (NC) subjects, and then systematically evaluated the diagnostic significance of both types of serum fluorescence alone and combined with AFP, hepatic function tests and/or conventional laboratory blood tests for diagnosing PHC, particularly early (BCLC stage A), small (tumor size ≤ 3 cm) and AFP-negative PHCs. Here, AFP is linked to cyclic hematopoiesis.