An electrochemical biosensor for the voltammetric detection of DNA sequences related to herpes simplex viruses (HSV) and discrimination of HSV Type I and Type II viruses from polymerase chain reaction (PCR) amplified real samples were described in this study. The biosensor relies on the covalent immobilization of the 22-mer single stranded oligonucleotides (probe) related to both HSV Type I and Type II sequences and hybridization of these oligonucleotides with their complementary and four bases mismatch containing (four bases MM) sequences at pencil graphite electrodes (PEGE). The extent of hybridization between probe and target sequences was determined by using differential pulse voltammetry (DPV) and Meldola Blue (MDB) was used as the hybridization indicator. As a result of the interaction between MDB and DNA at PEGE surface, the MDB signal observed from probe sequence before hybridization and after hybridization with four bases MM sequence is lower than that observed after hybridization with complementary sequence. The difference between the MDB signals obtained from probe modified, hybrid modified and four bases MM modified PEGE were used to detect and discriminate two types of HSV from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity. (C) 2004 Elsevier B.V. All rights reserved.