A squash print reverse transcription loop-mediated isothermal amplification amplification (SP-RT-LAMP) assay was developed for detection of Potato leafroll virus (PLRV) in a single aphid. Healthy aphids were given an acquisition feeding on potato plants infected with PLRV, and the acquired aphids were used for RNA isolation. The RT-LAMP assay was carried out using LAMP primers targeting the coat protein gene of PLRV. The amplified product was run on agarose gel; a typical ladder-like pattern in the acquired aphids indicated that the assay was able to detect PLRV from an individual aphid. The results of the RT-LAMP assay for the detection of PLRV in a single aphid were also visualized directly by adding fluorescent dye in LAMP amplified product. These results were confirmed by RT-PCR detection where an amplicon of expected size of 492 bp was observed in virus-acquired single aphids. The assay was optimized with respect to its isothermal amplification temperature and its incubation period. The technique was examined for its relative selectivity with other potato viruses transmitted by aphids where it did not show any cross-reactivity with the tested viruses indicating its specificity. With respect to its relative sensitivity, it was found to be equally sensitive as the existing RT-PCR assay. It was also found to be robust and highly reproducible as it was able to detect PLRV from known and unknown single aphid samples. The assay was successful in detection of PLRV in potato plants and tubers as well. Hence, we conclude that the assay is simple, sensitive and economical for detection of PLRV in single aphid and in potato plants and tubers.