Nucleic acid testing (NAT) based methods are more sensitive and specific, and are preferred over enzyme immunoassays. Different NAT based protocols have been designed to detect multiple pathogens in order to reduce the inherent high cost for detection. However, these assays do not reliably detect a large number of pathogens at once. In this report, a DNA hybridization based chemiluminescence detection method has been proposed for reliable detection of multiple pathogens. The idea was practically demonstrated by carrying out simultaneous extraction and amplification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 group M, herein referred to as HIV) through the one step multiplex reverse transcription polymerase chain reaction (RT-PCR). A detailed study was conducted to optimize the factors which could affect the chemiluminescence signal. The probes for HBV, HCV, and HIV demonstrated higher specificity by only capturing their respective target sequence. 10 viral copies per mL of serum were detected in the monoplex detection protocol for HBV, HCV, and HIV, respectively. However, when the sensitivity of each virus was analyzed in the presence of higher loads of other viruses in the multiplex detection assay, the assay finally detected 10 HBV copies, 10 HCV copies, and 100 HIV copies per microliter of serum. The use of silica modified magnetic nanoparticles during nucleic acid extraction and carboxyl coated magnetic nanoparticles during the chemiluminescence step can help modify this system into an automated platform for high throughput applications.