Femtosecond laser-assisted conjunctival autografts (CAG) preparation was recently proposed. This study reports the outcomes of the first clinical trial on the use of laser to prepare CAG in pterygium surgery, and to compare the outcomes with those of manual technique. Forty eyes undergoing primary pterygium excision with laser-assisted CAG transplantation were prospectively included (L group). Two historical matched cohorts whose CAGs were prepared manually were compared (n = 78 eyes by the same experienced surgeon, M group; n = 78 eyes by trainees; TM group). We found the laser-created CAGs had only 11 μm deviation from the targeted thickness. The best-corrected visual acuity improved, and the astigmatism significantly decreased after surgery, with comparable efficacy across 3 groups. The 1-year recurrence rate was 2.5%, 3.8% and 7.7% in the L, M and TM groups, respectively (P = 0.12). There was no significant difference between the L and M groups in the complication rate (5.0% and 1.3%, respectively), surgical time (19.4 ± 5.1 and 19.1 ± 6.2 minutes, respectively), and postoperative discomfort scores (0.1 ± 0.3 and 0.2 ± 0.3, respectively), but these outcomes were significantly less favorable in the TM group. The results of this first comparative clinical trial suggest that femtosecond laser-assisted CAG preparation can be considered as an alternative technique for CAGs preparation.