The need for a continuous, real-time monitoring of specific diseases represents an unmet scientific need. Evidently, cancer is one of the most important diseases where it is crucial to increase the rates of patient survival and monitor disease prognosis. Herein, a novel type of immunoassay was developed for detection of cancer biomarkers, using alpha-fetoprotein (AFP) and mucin-16 (MUC16) as model analytes. Using gold nanoparticle (AuNP) bioconjugates as a signal production tool, relevant antibody (Ab)-conjugated AuNPs were prepared on the nitrocellulose (NC) membrane. To construct a spot-like point-of-care (POC) immunoassay, cysteamine conjugated AuNPs (AuNP-Cys) were immobilized on the NC membrane and antibodies were conjugated to the nanoparticle on the detection pad, following a treatment with the samples that contains AFP or MUC16 which are well-known protein biomarkers for liver and ovarian cancer. By using the change in the colorimetric properties of AuNPs, detection of tumor markers was achieved by using a smartphone image and color analysis software at the final stage. Image J application was used for the evaluation of color changes depending on the biomarker concentration in buffer or spiked synthetic serum samples. The linear range was found as 0.1 ng/mL-100 ng/mL for AFP and 0.1-10 ng/mL for MUC16. Limit-of-detection (LOA) was calculated as 1.054 ng/mL and 0.413 ng/mL for AFP and MUC16, respectively. Interferent molecules, Her2, Immunoglobulin G (IgG) and bovine serum albumin (BSA) were tested on the system. Furthermore, synthetic serum samples spiked with selected analyte molecule were applied on the system and measured successfully.