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The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, <i>Dendrobium officinale</i>, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the <i>D. officinale</i> genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all <i>DoAP2</i> genes contained <i>cis</i>-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of <i>DoAP2-2</i>, <i>DoAP2-5</i>, <i>DoAP2-7</i>, <i>DoAP2-8</i> and <i>DoAP2-12</i> genes in protocorm-like bodies (PLBs), while <i>DoAP2-3</i>, <i>DoAP2-4</i>, <i>DoAP2-6</i>, <i>DoAP2-9</i>, <i>DoAP2-10</i> and <i>DoAP2-11</i> expression was strong in plantlets. In addition, the expression of some <i>DoAP2</i> genes was down-regulated during flower development. These results suggest that <i>DoAP2</i> genes may play roles in plant regeneration and flower development in <i>D. officinale</i>. Four <i>DoAP2</i> genes (<i>DoAP2-1</i> from euAP2, <i>DoAP2</i>-<i>2</i> from euANT, and <i>DoAP2-6</i> and <i>DoAP2-11</i> from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of <i>Arabidopsis thaliana</i> mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in <i>Nicotiana benthamiana</i> leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on <i>AP2</i> genes in <i>D. officinale</i> shed light on the function of <i>AP2</i> genes in this orchid and other plant species.