Tylenchulus semipenetrans is an economically important plant-parasitic nematode occurring in all citrus-producing regions of the world and causing a disease called "slow decline". For the rapid detection of this nematode, a loop-mediated isothermal amplification (LAMP) assay was developed, based on the ribosomal DNA internal transcribed spacer sequence. The optimal condition for the LAMP assay was 65°C for 50 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR) and restriction analysis with the BamHI enzyme, and by adding SYBR Green I to the LAMP products for visual inspection. The LAMP assay was highly specific for the detection of T. semipenetrans populations from different geographical origins. It was also sensitive, detecting a tenth of the DNA from an individual specimen of T. semipenetrans, which was 10 times more sensitive than conventional PCR. The LAMP protocol was applied to natural citrus rhizosphere soil samples from several orchards in China and the results were fast, sensitive, robust, and accurate. This study is the first to provide a diagnostic tool for T. semipenetrans using DNA extracted directly from citrus rhizosphere soils. This LAMP assay could be used as a practical molecular tool to identify T. semipenetrans and diagnose slow decline disease, even in remote locations.