Background: To evaluate the initial safety and efficacy of femtosecond laserassisted minimal invasive lamellar keratoplasty (FL-MILK) for advanced keratoconus. Methods: Twenty-two patients (22 eyes) with advanced keratoconus were included in this prospective study. All the involved eyes underwent FL-MILK.The femtosecond laser was used to create an intrastromal pocket with a 2.3 mm incision in the recipient cornea. Then a stromal button with a diameter of 9.0 mm and a depth of 200 μm was gently inserted into the intrastromal pocket through the 2.3 mm incision and flattened. No sutures were applied.Follow-up was conducted for 24 months.Results: Twenty-two patients completed follow-up data for 12 months, 16 patients had 24 months follow-up. No epithelial implantation, infection or allogeneic rejection were observed during the follow-up. Based on baseline values, postoperative 12 months values and postoperative 24 months values, clinical significantly improvement was recorded in corrected distance visual acuity (CDVA) (0.40 ± 0.18 logMAR vs. 0.30 ± 0.12 logMAR and 0.23 ± 0.13 logMAR), the anterior central corneal elevation (29.14 ± 15.33 μm vs. 14.45 ± 13.75μm and 11.38 ± 8.33 μm), and corneal higher-order aberrations (3.536 ± 1.503 vs. 2.761 ± 1.517 and 0.994 ± 0.391). Corneal biomechanical properties in all eyes improved significantly. SP-A1 increased from 48.64 ± 12.87 preoperatively to 87.26 ± 21.01 postoperative 12 months and 88.77 ± 18.26 postoperative 24 months; deformation amplitude (DA) decreased from 1.36 ± 0.15 preoperatively to 1.21 ± 0.12 postoperative 12 months and 1.19 ± 0.19 postoperative 24 months.Conclusions: Initial experience suggests that this minimally invasive transplantation may be a feasible option for advanced keratoconus. A larger cohort and longer follow-up are required to validate our results and establish longterm safety and efficacy of the procedure.Hua Gao and Weiyun Shi contributed equally to this manuscript.