We investigated the predictive biomarkers for graft rejection in pig‐to‐non‐human primate (NHP) full‐thickness corneal xenotransplantation (n = 34). The graft score (0‐12) was calculated based on opacity, edema, and vascularization. Scores ≥ 6 were defined as rejection. NHPs were divided into two groups: (a) graft rejection within 6 months; and (b) graft survival until 6 months. In the evaluation of 2‐week biomarkers, none of the NHPs showed rejection within 2 weeks and the 34 NHPs were divided into two groups: (a) entire rejection group (n = 16); and (b) survival group (n = 18). In the evaluation of 4‐week biomarkers, four NHPs showing rejection within 4 weeks were excluded and the remaining 30 NHPs were divided into two groups: (a) late rejection group (n = 12); and (b) survival group (n = 18). Analysis of biomarker candidates included T/B‐cell subsets, levels of anti‐αGal IgG/M, donor‐specific IgG/M from blood, and C3a from plasma and aqueous humor (AH). CD8+IFNγ+ cells at week 2 and AH C3a at week 4 were significantly elevated in the rejection group. Receiver operating characteristic areas under the curve was highest for AH C3a (0.847) followed by CD8+IFNγ+ cells (both the concentration and percentage: 0.715), indicating excellent or acceptable discrimination ability, which suggests that CD8+IFNγ+ cells at week 2 and AH C3a at week 4 are reliable biomarkers for predicting rejection in pig‐to‐NHP corneal xenotransplantation.