Flavonoids are the major medicinally active ingredients that exert potential effects in Amomum tsao-ko. In total, 277 flavonoid metabolites were identified in fresh and dried fruits of three different accessions of A. tsao-ko (Amomum tsao-ko), which could be classified into eight classes with more metabolites classified as flavonol. Furthermore, 193 differential flavonoid metabolites (DFMs) were selected from the six samples (three fresh and dried fruits), the main DFMs identified in the fresh and dried fruits of A. tsao-ko were apigenin and its derivatives, and 11 main DFMs were identified. Upon analyzing the variations in flavonoid content between fresh and dried fruits, our findings indicate that the L accession demonstrate a superior accumulation of flavonoid metabolites in their dried fruits. Combination with transcriptome data, the synthesis of flavone and isoflavone metabolites in fresh fruits of A. tsao-ko may be affected by the changes in the expression of chalcone isomerase genes and regulated by the NAC and AP2 transcription factor. This study provides an important theoretical basis for the functional study of flavonoid synthesis-related genes and transcription factors in A. tsao-ko fruits and for the breeding of these fruits with high flavonoid content.