In this study, we present a strategy for the specific electrochemical detection of NADH in human blood serums using modified electrode fabricated by intercalation of tetrabutylammonium ions (TBA(+)) into pencil graphite electrode (TBA(+)/PGE). In order to implement this protocol, a modification process in the potential range (-0.2) to (-2.75) V was performed in DMSO containing 0.1 M TBAP by CV method. The characterization processes of the prepared sensor were carried out using CV, EIS, FT-IR, XPS, and SEM methods. The electrode fabricated by intercalation of TBA(+) was found to be able to analyze NADH at +0.32 V applied potential by the amperometric method. The LOD was detected as 0.46 mu M. The analysis results of human blood serums which are taken from healthy individuals show that TBA(+)/PGE can be used for real samples. The results indicated that TBA(+)/PGE can be easily fabricated by one-step and one-pot electrochemical method and TBA(+)/PGE can be used as a potential sensor for the NADH determination.