The detection of foodborne pathogens is critical for disease control and infection prevention, especially in seafood consumed raw or undercooked. Paper-based diagnostic tools are promising for rapid fieldable detection and provide a readout by eye due to the use of gold nanoparticle immunoprobes. Here we study different strategies to overcome these challenges in a real biological matrix, oyster hemolymph, for the detection of the pathogenic bacteria Vibrio parahaemolyticus (Vp). Nanoparticle surface chemistry, nitrocellulose speed and blocking, running steps, and antibody concentrations on the NP and nitrocellulose were all studied. Their effect on paper immunoassay signal intensity was quantified to determine optimal conditions, which enabled the detection of Vp directly from hemolymph below pathogenic concentrations.