The sunblotch disease is one of the diseases that affects avocado (Persea americana Mill) crops in particular, causing serious crop losses. The causal agent is the avocado sunblotch viroid (ASBVd), a circular RNA-based pathogen built of 247 nucleotides, which causes stem discoloration, bleaching, variegation, and distortion on the leaves and sunken yellow or red blotches on the fruits. In this study, a novel SYBR green-based real-time RT-PCR (RT-qPCR) assay specific for ASB viroid was developed. The assay was followed by a standard curve and a melting curve analysis for the detection, identification, and quantification of the full-length ASB viroid. The assay’s lower limit of detection in the samples of avocado leaves was 8.8 copies/μl. The RT-qPCR was 100 times more sensitive to ASBVd than the conventional RT-PCR. The melting curve analysis showed a variation in the specific melting temperature (Tm) between positive samples, probably due to the alteration in the nucleotide sequences of viroids. In conclusion, the use of RT-qPCR assays established in this study, as a routine diagnostic method in breeding programs, could decrease the viroid incidence in avocado crops and its further accumulation in plants.