[Background] Acinetobacter baumannii is highly resistant to antibiotics. Gene knockout is an important way to study the virulence and antibiotic resistance of A. baumannii. Currently, most gene knockout methods rely on the use of an antibiotic selection marker and are not suited for multidrug resistant strains. [Objective] This study aims to establish a marker-less gene deletion method to knock out the genes of multidrug resistant A. baumannii for subsequent experiments. [Methods] We used a two-step screening method using homologous recombination and the resistance of pMo130-TelR to potassium tellurite to construct the type VI secretion system hcp gene deletion mutant of A. baumannii. Then we tested the growth ability, bacterial competition ability and serum resistance of the mutant strain. [Results] By constructing a tellurite-resistant suicide vector inserted with recombinant fragment, pMo130-TelR-(hcp up-down), we successfully knocked out the hcp gene in A. baumannii. There were no significant changes in the growth ability of mutants, in contrast, the bacterial competitive ability decreased and the serum resistance increased significantly. [Conclusion] The marker-less gene deletion method using pMo130-TelR is applicable for creating gene deletion mutants in A. baumannii. It will be very useful in multidrug resistant A. baumannii gene deletion and will have profound significance for A. baumannii research such as the antibiotic-resistance mechanism.