Background: Opaque bubble layer (OBL), which generates from photo-disruptive procedures on the cornea, has been a common phenomenon during femtosecond laser-assisted refractive surgeries and it would potentially impact eye tracking and flap lifting. And we have observed that an updated flap-making pattern could form less OBL clinically than the traditional pattern, which needed further approval. Thus, the purpose of this study is to prove our observation and investigate the possible risk factors related to the occurrence and type of OBL in laser in situ keratomileusis (LASIK) flaps using the Visumax laser system. Methods: This prospective study included 167 eyes of 86 patients (mean age: 27.5 +/- 6.1 years) undergoing bilateral femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK) for myopia/myopic astigmatism by the same surgeon from April 2020 to August 2020. Preoperative data on refraction, central corneal thickness (CCT), and keratometry as well as intraoperative data were included for analysis. A new flap-making pattern creating an offset between flap-cut and side-cut was adopted to compare with the traditional pattern. The operation video of flap formation was analyzed to identify the existence and type of OBL. The area covered by OBL and the ratio of OBL to flap were calculated using Image J software. Results: Among 167 eyes, 54 eyes (32.3%) developed OBLs, consisting of 31 as hard OBL coexisting with soft OBL, and 23 as soft OBL alone. The OBL incidence was significantly reduced in eyes with the new flap-making pattern compared with the traditional pattern (13.8% vs. 52.5%, P < 0.001). Hard OBLs had larger area ratios than soft OBLs (14.3 +/- 8.3% vs. 1.1 +/- 1.8%, P < 0.001). Univariate analyses revealed that eyes with more myopia, thicker CCT, and traditional flap-making patterns were more likely to develop OBLs. Multivariate analysis further confirmed that more myopia, thicker CCT, and traditional flap-making pattern were risk factors for OBLs. A Larger corneal diameter was associated with a higher incidence of hard OBL when applying the traditional flap-making process. Conclusion: More myopia, thicker CCT, and larger corneal diameter were risk factors for OBL development during flap creation, whereas a flap-making pattern with an offset between flap-cut and side-cut could reduce the incidence of OBL.