A new spectrofluorimetric method has been developed for the quantitative determination of the alpinetin content in cell culture medium samples. This method combines excitation-emission matrix fluorescence with chemometric second-order calibration methods based on Parallel Factor Analysis(PARAFAC) and Self-weighted Alternating Trilinear Decomposition (SWATLD) algorithms. When the component number was chosen to be 2, the average recoveries obtained were (100.3±2.2) % for PARAFAC and (100.1±2.2) % for SWATLD, respectively. The limits of determination for alpinetin were 0.182 3 μg·mL-1 for PARAFAC and 0.178 1 μg·mL-1 for SWATLD. EJCR has demonstrated that the results are accurate.