Maricaulales, the taxonomic Latin nomenclature for a particular bacterial order, have emerged as a prominent group among marine dimorphic prosthecate bacteria, primarily due to their distinctive cellular morphology. This study selected four representative strains of to devise a standardized gene knockout methodology. Through a systematic optimization of conjugation transfer factors, a dependable conjugation transfer technique was successfully established. Following this, the utilization of the optimized conjugation transfer protocol facilitated the successful knockout of the genes encoding the holdfast anchor protein in all four chosen Maricaulales strains. In conclusion, this research introduces a novel and efficient gene knockout tool specifically developed for engineering Maricaulales.