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Background Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes a zoonotic disease commonly called Q fever globally. In this study, an up-converting phosphor technology-based lateral flow (UPT-LF) assay was established for the rapid and specific detection of phase I strains of C. burnetii . Results Specific monoclonal antibodies (10B5 and 10G7) against C. burnetii phase I strains were prepared and selected for use in the UPT-LF assay by the double-antibody-sandwich method. The detection sensitivity of the Coxiella -UPT-LF was 5 × 10 4 GE/ml for a purified C. burnetii phase I strain and 10 ng/ml for LPS of C. burnetii Nine Mile phase I (NMI). Good linearity was observed for C. burnetii phase I and NMI LPS quantification (R 2  ≥ 0.989). The UPT-LF assay also exhibited a high specificity to C. burnetii , without false-positive results even at 10 8 GE/ml of non-specific bacteria, and good inclusivity for detecting different phase I strains of C. burnetii . Moreover, the performance of the Coxiella -UPT-LF assay was further confirmed using experimentally and naturally infected samples. Conclusions Our results indicate that Coxiella -UPT-LF is a sensitive and reliable method for rapid screening of C. burnetii, suitable for on-site detection in the field.