Onion (Allium cepa L.) is a major vegetable used in food preparation. It can also act as a medicinal compound for many diseases. However, various plant diseases, including sour rot, caused by the bacterium Burkholderia cepacia, is a post-harvest disease that can cause significant economic losses in onions and are considered a major threat to the onion industry. This study was performed to compare two methods for detecting sour rot: multiplex PCR and colorimetry with gold nanoparticles probe. Multiplex PCR used, primers designed for RecA, APZ15-03995 and APZ15-27735 genes of B. cepacia, and the results showed 100% specificity and sensitivity of 74.4 × 10−4 ng/μL of genomic DNA. Colorimetry with gold nanoparticles probe was performed using individual gene probes or a combination of three gene probes as a multi-colorimetric assay. Results showed 100% specificity and sensitivity of 74.4 × 10−8 ng/μL of genomic DNA in presence of the three probes. The results showed that both methods are specific and sensitive enough to detect B. cepacia. However, the use of gold nanoparticles is a more suitable option due to its high specificity and sensitivity and shorter time compared to multiplex PCR. Consequently, accurate and timely molecular identification of onion rot helps reduction of post-harvest losses and prevent the spread of secondary bacteria in storage.