The invention relates to a nucleic acid reagent, a kit, a system and a method for detecting Gram-negative bacteria and/or drug resistance of Gram-negative bacteria. The nucleic acid reagent comprisesprimers shown as the SEQ ID NO. 1-60 and probes shown as the SEQ ID NO. 63-92 which are independently stored or stored as arbitrary mixtures. By using the primers and the probes, the nucleic acid reagent, the kit, the system and the method for detecting 6 pathogens, including Klebsiella pneumoniae, Escherichia coli, pseudomonas aeruginosa, enterobacter cloacae, serratia marcescens and acinetobacter baumannii, as well as 24 common drug resistance genes carried by the 6 pathogens are established by the invention; moreover, fast, comprehensive, sensitive, specific and automatic detection result determination can be realized, so that, sensitivity, specificity and simplicity of simultaneous detection of multiple Gram-negative bacteria and drug resistance thereof are significantly improved.