The invention discloses a Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method. The detection kit comprises a PCR reaction solution, an enzyme mixed solution, a positive control, a negative control and an internal standard, wherein the PCR reaction solution comprises a PCR buffer solution, nucleic acid releaser, deoxyribonucleoside triphosphate, primers for target polynucleotide amplification and probes for target polynucleotide detection; the primers comprise a forward primer and a reverse primer. Or, the detection kit comprises a PCR reaction solution, an enzyme mixed solution and an internal standard, wherein the PCR reaction solution comprises a PCR buffer solution, nucleic acid releaser, deoxyribonucleoside triphosphate, a forward primer for target polynucleotide amplification, a reverse primer for target polynucleotide amplification and probes for target polynucleotide detection. The Pseudomonas aeruginosa nucleic acid fluorescent PCR detection kit shown in the embodiment of the invention with the advantages of no need of nucleic acid extraction, high detection sensitivity, wide detection range and high detection accuracy can quickly and accurately detect Pseudomonas aeruginosa DNA (deoxyribonucleic acid) in plasma, urine and other samples.