An efficient genome editing tool, the CRISPR/Cas12a system, has been used in research on plant functional genomics and the improvement of agronomic traits. In this study, the CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. With this system, crRNAs were driven by the Pol Ⅱ pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation. The vector worked efficiently in all positive transgenic plants and the editing efficiencies at the crRNA1 and crRNA2 target sites were up to 93.37 and 88.24%, respectively. This system had significantly higher editing efficiency than the pGhRBE3 system with a Pol Ⅲ promoter-Ubi 6.7 promoter, indicating that the Pol Ⅱ promoter is more suitable for expressing multiple sgRNAs or crRNAs than the Pol Ⅲ promoter in cotton. The vector mainly generated the editing type of fragment deletion, and the deletion sizes were in the range of 3–12 bp with the editing sites spanning the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from the T₀ to T₂ generations, and three transgene-free lines with target site mutations in the GhPGF gene were obtained. These glandless and gossypol-free (or low content) cotton germplasms will play a key role in healthy cottonseed oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, so it can provide a powerful tool for cotton functional genomics and genetic improvement.