This study aimed to establish an efficient and precise cytosine base editor (CBE)-mediated knockout system in chicken somatic cells and primordial germ cells (PGCs). PGCs are pivotal for generating genome-edited chickens, but low transfection efficiency limit their application. Unlike CRISPR/Cas9, CBEs achieve precise C-to-T conversion without DNA double-strand breaks or donor templates, making them safer for avian genome engineering. We used CBEs to introduce premature stop codons in exon 1 of the sex-determining AR and DMRT1 genes for targeted knockout. Among 12 screened sgRNAs, sgRNA6 (AR, 94.67 ± 6.66%) and sgRNA9 (DMRT1, 6.67 ± 6.51%) performed best in DF-1 cells; in PGCs, their editing efficiencies reached 51.0% and 91.0%, respectively. No off-target mutations were detected in edited DF-1 cells. These findings confirm that CBE-mediated knockout is highly efficient and safe in chicken somatic and germ cells, providing a robust tool for avian genome editing.