Based on the third generation genome of longan (Dimocarpus longan), we isolated the cDNA sequence of AGO4 gene from longan embryogenic callus (cultivar:‘Honghezi’) by reverse transcription polymerase chain reaction (RTPCR) and analyzed the bioinformation. Furthermore, we analyzed the expression profiles in somatic embryogenesis, different tissues, hormones treatment, abiotic stress, and 5azac (5azacytidine) treatment. The results showed that: (1) The cDNA length of DlAGO4 was 3 425 bp, which contained an open reading frame of 2 781 bp and encoded 926 amino acids. (2) The bioinformatic analysis showed that it was basic, hydrophilic and nonsecretory protein, containing PAZ and Piwi conserved domains, had 85 phosphorylation sites and 2 glycation sites, while shared high homology with CcAGO4 of Citrus clementina. The subcellular localization was predicted to be located in the nucleus, and microRNA prediction showed that DlAGO4 was regulated by four miRNAs. (3) qRTPCR results indicated that the relative expression of DlAGO4 was upexpressed in the globular embryos (GE) and seeds in Longan, while KT, salicylic acid, NaCl, mannitol, PEG4000 and ABA treatments could promote the expression, but 2,4D and MeJA treatments inhibited the expression. After treated with 5azac, the expression of DlAGO4 was downregulated in cultured 1 d and 3 d, but upregulated from cultured 6 d, and significantly increased in cultured 12 d. These results suggested that DlAGO4 might be involved in the transcriptional regulation of globular embryo stages and seeds development of longan. Furthermore, DlAGO4 might be involved in hormone, abiotic stress responsiveness and DNA methylation regulation.