In　this　paper,　a　sensitive　and　specific　competitive　fluorescence　immunoassay　(CFIA)　method　was　developed　for　the　qualitative　and　quantitative　analysis　of　ketamine　(KET).　A　novel　competitive　model　in　which　ketamine　hapten　(KET-BSA),　coated　on　microporous　plates,　competed　with　ketamine　antigen　(KET-Ag)　in　actual　samples　to　bind　fluorescein　isothiocyanate-labeled　antibody　(KET-Ab)　could　be　used　for　rapid　and　indirect　quantitative　analysis　of　KET　in　human　urine,　blood,　or　sewage.　In　the　CFIA　method,　KET　concentration　in　the　sample　negatively　correlated　with　the　detected　fluorescence　intensity.　The　linear　correlation　coefficient　of　the　competitive　quantitative　equation　was　0.992,　the　linear　range　was　0.01-0.5　μg　mL−1,　and　the　limit　of　detection　(LOD)　was　0.1　pg　mL−1.　The　specificity　results　showed　that　the　cross-reaction　rate　of　norketamine　was　less　than　10%.　Recoveries　of　spiked　samples　at　low,　medium,　and　high　concentrations　ranged　from　96%　to　117%.　The　CFIA　method　and　classical　gas　chromatography-tandem　mass　spectrometry　(GC-MS/MS)　were　used　to　detect　the　actual　samples　simultaneously.　The　relative　deviation　of　the　quantitative　results　was　less　than　10%.　The　LOD　value　of　KET　by　CFIA　was　four　orders　of　magnitude　lower　than　that　by　GC-MS/MS.　Additionally,　CFIA　had　great　advantages　over　GC-MS/MS　in　terms　of　sample　pretreatment　and　economic　investment.　In　conclusion,　this　study　provided　a　targeting　detection　platform　for　KET,　which　achieved　a　rapid,　portable,　and　sensitive　analysis　of　trace　KET　in　various　materials.　©　2022　The　Royal　Society　of　Chemistry.