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To describe the feasibility and outcomes of intraoperative optical coherence tomography (OCT)–guided, femtosecond laser–assisted deep anterior lamellar keratoplasty in which the lamellar and tunnel cuts were completed by laser.The Ziemer Z8 system was used to scan the corneas intraoperatively and guide the stromal lamellar and tunnel cuts created by the laser. Thirty porcine eyes and 3 human cadaver globes were used to evaluate the accuracy of laser-created intrastromal tunnels and the feasibility of achieving big-bubble pneumatic dissection. The tunnel distance above the Descemet membrane (Dt-dm) was set at 100 and 130 μm, and the accuracy of laser cut was assessed by anterior segment OCT and histological sections. Fourteen patients with keratoconus or corneal scars were then included to evaluate the clinical outcomes.The deviation from the targeted Dt-dm was 5.5% to 8.6% and 0.7% to 9.8% for the 100 and 130 μm groups, respectively. The achieved thickness had a significantly and positively strong correlation with the targeted thickness (r = 0.86; P < 0.001). In all cases, the anterior stromal lamella was removed without difficulty, except in the patients with corneal scars in whom some remaining stromal fiber attachments were observed. A type 1 big bubble was successfully achieved in all patients.We present the accuracy and clinical outcomes of femtosecond laser–assisted, intraoperative OCT–guided deep anterior lamellar keratoplasty, in which controlled lamellar and tunnel cuts at desired depths were performed by laser in one step, and a successful big bubble was achieved.