The invention provides a detection kit for human sepsis pathogens and a corresponding detection method. The kit of the present invention includes detection of Acinetobacter baumannii, Klebsiella pneumoniae, Streptococcus pneumoniae, Bacteroides fragilis, Staphylococcus aureus, Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli bacteria, Enterococcus gallinarum, Enterobacter cloacae, Stenotrophomonas maltophilia, Coagulase-negative Staphylococcus, Candida albicans, Candida tropicalis, Candida glabrata, Cytomegalovirus, Human herpesvirus type IV , Human parvovirus B19, Candida parapsilosis, Streptococcus primers and fluorescent probes.