Purpose: To investigate clinical characteristics and tear film biomarkers of patients with chronic dry eye disease (DED) following femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK). Methods: Patients were divided into the chronic DED after FS-LASIK (n = 36), DED without FS-LASIK (n = 39), and normal control (without FS-LASIK; n = 34) groups. Dry eye, pain, and psychological-related symptoms were evaluated using the Ocular Surface Disease Index (OSDI), Numerical Rating Scale (NRS), Neuropathic Pain Symptom Inventory Modified for the Eye (NPSI-Eye), and Hamilton Anxiety Rating Scale (HAMA) questionnaires. Ocular surface parameters, tear cytokines, and neuropeptide concentrations were evaluated with specific tests. Results: The DED after FS-LASIK group showed higher corneal fluorescein staining scores, but lower OSDI and NPSI-Eye scores than the DED without FS-LASIK group (all P < .05). Corneal sensitivity and nerve density decreased in the DED after FS-LASIK group (all P < .01). Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-17A, IL-23, alpha-melanocyte stimulating hormone (α-MSH), oxytocin, and substance P levels were highest in the DED after FS-LASIK group, followed by the DED without FS-LASIK and normal control groups (all P < .05). Interferon-γ and neurotensin levels were only significantly higher in the DED after FS-LASIK group (all P < .05). Conclusions: Patients with chronic DED after FS-LASIK showed milder ocular symptoms, greater epithelial damage, and higher levels of tear inflammatory cytokines and neuropeptides than patients with DED without FS-LASIK, indicating that the nervous and immune systems may play significant roles in FS-LASIK-related chronic DED development.