Rapid detection of trace mercury (II) in water is very challenging. A novel sensor for the facile detection of mercury (II) was developed using electrochemically co-deposited gold nanofilm modified screen-printed carbon electrodes (EcoD-AuNF/SPCE). Because a monolayer of gold nanofilm is freshly co-deposited during the mercury (II) preconcentration period, no additional modification on an electrode is required prior to a test. Central composite design (CCD) and response surface methodology (RSM) were used to evaluate the effects of the critical testing parameters simultaneously (i.e., the co-deposited gold concentration of $350\,\,\mu \text{g}\,\,\text{L}^{-1}$ , deposition potential of −0.75 V, and testing pH of 3.50). The response of square-wave anodic stripping voltammetry (SWASV) showed a linear relationship with the mercury (II) concentration over a range of 1 to $40~\mu \text{g}$ /L (ppb), with an LoD of $0.29~\mu \text{g}$ /L and sensitivity of $0.37~\mu \text{A}$ /ppb. Significant influences on the mercury (II) stripping peak potential, peak height, and peak shape were observed in the presence of chloride. Therefore, an optimized amount of chloride (i.e., 600 mg/L) was preemptively added to minimize the potential effect of naturally occurring chloride and to improve further the detection sensitivity (i.e., LoD of $0.16~\mu \text{g}$ /L and sensitivity of $0.55~\mu \text{A}$ /ppb). Validation testing using real-world water samples indicated reliable prediction of mercury (II) concentration could be obtained in complex media. In summary, this newly developed voltammetric approach has excellent detection performance and practical significance for potential on-site voltammetric determination of trace mercury (II) in water.