Purpose: The purpose of this study was to analyze tear cytokine and complement levels in patients diagnosed with acute ocular graft-versus-host disease (oGVHD) and examine the consistency of these levels with the severity of clinical manifestations. Methods: Ten patients with acute oGVHD (20 eyes) were enrolled for the assessment of tear cytokine levels and ocular surface parameters, and 18 healthy people (36 eyes) were selected as the control group. The tear cytokine and complement levels were measured using microsphere-based immunoassay analysis. Results: The main clinical manifestations of acute oGVHD include eye redness, a large amount of purulent exudate, eye pain, and even false membranes. The levels of intercellular cell adhesion molecule-1, interleukin 6 (IL-6), interleukin 1 beta (IL-1b), interleukin 8, epidermal growth factor (EGF), interleukin 7 (IL-7), B-cell activating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and complement in patients with acute oGVHD showed significant differences compared with those in normal people. Furthermore, the levels of IL-6, IL-1b, EGF, GM-CSF, IL-7, and C3a showed a stronger correlation with ocular surface parameters. Conclusions: Our study was the first to enroll patients with acute oGVHD to assess tear cytokine levels as a method contributing to the diagnosis of acute oGVHD. In addition, it has been demonstrated that certain tear cytokines, including intercellular cell adhesion molecule-1, IL-6, IL-1b, interleukin 8, B-cell activating factor, GM-CSF, IL-7, EGF, and complement, may be new diagnostic biomarkers for acute oGVHD.