Over the last decade, many applications of electrochemical sensors/biosensors have been developed with various considerations such as cost-effectiveness, process simplicity, high sensitivity and accuracy, the need for the amount of analyte, and a miniature structure with portability. Until now, various electrochemical methods have been used to develop biosensors for detecting molecular markers, especially their modifications with nanoparticles. Historically, gold is a very inert material with little or no reactivity; thus, nanosized gold particles have been shown to function as an effective catalyst for a number of chemical reactions under various experimental conditions. In this study, the design of experiment (DoE) is used to create a model and analyze the best response that is affected by several independent variables with the aim of optimizing the response. The results showed that gold nanoparticles (AuNPs) were able to increase the peak current on the screen-printed carbon electrode-gold nanoparticle electrode and produced two of the most significant factors, namely, the concentration of single-stranded deoxyribonucleic acid (ssDNA) probe and the attachment time of AuNPs.