In this study, the EjAGL6 gene was cloned from the flower buds of ‘Huayuwuhe No.1’ in triploid loquat (Eriobotrya japonica) by gene cloning, and its sequence, subcellular localization and expression level in diploid and triploid loquat cultivars were analyzed. Arabidopsis thaliana was infected by flower dipping method. The expression level of EjAGL6 gene in transgenic A. thaliana plants was analyzed by qRTPCR. The phenotypic differences between wildtype A. thaliana and EjAGL6 transgenic A. thaliana were observed, and the function of EjAGL6 gene was analyzed, providing theoretical basis for analyzing the mechanism of EjAGL6 gene involved in triploid loquat flowering. The results show: (1) Madsbox gene EjAGL6 was obtained successfully. The encoding region (CDS) of EjAGL6 is 732 bp, encoding 243 amino acids. The molecular weight of the putative protein is 27.88 kD, isoelectric point is 9.05, and liposolubility index is 79.05. Phylogenetic tree analysis showed that EjAGL6 and MdAGL6 had high homology and clustered into the same branch. (2) Protein sequence alignment suggested that EjAGL6 includes M domain (57 aa), I domain (30 aa), K domain (82 aa) and the C domain (74 aa), and two highly conserved motifs of AGL6 motifs I and II in the Cterminal domains. (3) Subcellular localization analysis indicated that the functional region of EjAGL6 protein was located in the nucleus, suggesting it has subcellular localization characteristics of MADSbox transcription factor. (4) The qRTPCR analysis showed that EjAGL6 was expressed in diploid and triploid loquat early and late flowering cultivars. Among them, EjAGL6 expression level was mainly concentrated in the latter three stages of flower development, including the stages of florets (S6), white corollas of floral buds (S7) and full bloom (S8), and the EjAGL6 expression level of diploid and triploid early flowering cultivars were higher in white corollars stage of floral buds. (5) The expression level of EjAGL6 gene in transgenic A. thaliana was significantly higher than that in wildtype A. thaliana. Phenotype observation of transgenic plants showed that overexpression of EjAGL6 gene in A. thaliana could advance flowering time of transgenic A. thaliana by one week. The results showed that EjAGL6 gene could promote the flowering time of loquat earlier, suggesting that EjAGL6 gene plays a key role in regulating flowering time during white corollas stage of floral buds.