To explore the target relationship between hrhmiR319e and transcription factor AP4 of sea buckthorn, and analyze their expressional changes in developing seeds, we selected the seeds at different developmental stages as study materials and used RNA hybrid software to predict the binding sites in the 3′UTR region of the candidate target gene AP4 by miR319e seed mature sequence. The pcDNA3.1+pmirGLOAP4 and pcDNA3.1hrhmir319e+pmirGLOAP4 vectors were constructed respectively. The method of double luciferase reporter gene detection was used to verify whether miR319e could target the 3′UTR region of candidate target gene AP4, and the expression changes of miR319e and AP4 were analyzed using realtime fluorescent PCR technology in developing sea buckthorn seeds. The results showed that: (1) AP43′UTR region and miR319e seed mature sequence were completely complementary. (2) The plasmid vector of pcDNA3.1hrhmir319e+pmirGLOAP4 can significantly inhibit the luciferase activity (P< 0.001). (3) In developing seeds, the changes of hrhmiR319e expression level showed a trend of increasing at first and then decreasing, while the changes of AP4 gene expression level first decreased and then increased. When hrhmiR319e expression reached its highest level at 80 d after flowering, it was 3.146 in ‘Xine 3’, 4.298 in ‘Suiji 1’ and 3.892 in ‘Za 56’, respectively; but the expression level of AP4 gene had the lowest value, it was 0.427 in ‘Xine 3’, 0.526 in ‘Suiji 1’ and 0.451 in ‘Za 56’, respectively. Thus, sea buckthorn hrhmiR319e could target the 3′UTR region of AP4 gene, and there is negatively regulative relationship between them. These data provided scientific references for further understanding the seed development mechanism of sea buckthorn and the cultivation of large seed varieties.