In this study, a pretreatment protocol was developed, and combined with phthalaldehyde (OPA) and 9-fluorenyl methyl chloroformate (FMOC-Cl) pre-column derivatization, RP-HPLC method, and fluorescent detection for the quantitative and qualitative investigations of dissolved free amino acids (DFAA) in biogas slurry. Owing to the simultaneous removal of high-strength ammonium and biogenic amines by pH adjustment during rotary evaporation, the recovery of DFAA was greatly improved by the introduction of 3K Millipore ultrafiltration tubes. As a result, a good linear relationship (corresponding linear correlation coefficient (R²) greater than 0.99) was obtained with the spike recovery rate of 23 kinds of amino acid of 89%~115%, except for that of glutamic acid of 70.5%. And the relative standard deviation was 1.1%~5.0% for the recovery analysis. On this basis, the developed pre-treatment method was further carried out to different sludge samples. The DFAA was 2.0mol/L in the excess sludge, while the amount of DFAA in the biogas slurry with input TS of 5% and 20% were different in species and amounts with 0.04mol/L and 1.94mol/L, respectively. Thus, no correlation between the DFAA amount and the input had been observed, which proved that the generation and utilization mechanisms of amino acids varied in different anaerobic digesters.