<p>Acute graft-versus-host disease (aGVHD) remains a life-threatening complication that limits the efficacy and application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Early identification and diagnosis of aGVHD is significant for improving the outcomes of allo-HSCT. We retrospectively analyzed the dendritic cell (DC) subsets in peripheral blood (PB) of patients who received allo-HSCT before transplantation and at engraftment to evaluate the correlation between DC subsets and the occurrence of aGVHD. The results showed that the proportion and count of myeloid dendritic cells (mDCs) before transplantation were positively correlated with the occurrence and severity of aGVHD, while the proportion and count of mDCs at engraftment were negatively correlated with those. In addition, patients with higher proportion and count of mDCs before transplantation developed aGVHD earlier, whereas those without aGVHD had the lowest proportion and count of mDCs before transplantation. The proportions and counts of DC subsets at engraftment were not significantly correlated with the onset time of aGVHD. Receiver operating characteristic (ROC) curve analysis demonstrated that the count of mDCs in PB before transplantation had greater sensitivity and specificity in predicting aGVHD (<i>AUC</i> = 0.8002, <i>P</i> &lt; 0.0001) compared to plasmacytoid DCs (pDCs) and total DCs. Multivariate logistic regression analysis confirmed that the count of mDCs in PB before transplantation and at engraftment were independent factors in predicting the occurrence of aGVHD (<i>OR</i> = 9.907, <i>P</i> = 0.018, 95% <i>CI</i> 1.473–66.639; <i>OR</i> = 0.059, <i>P</i> = 0.023, 95% <i>CI</i> 0.005–0.673). Therefore, mDCs in PB play an important role in the development of aGVHD and may serve as a promising predictor of aGVHD for patients undergoing allo-HSCT.</p>