A novel nonseparation electrochemical enzyme immunoassay (NEEIA) for detecting marker proteins in undiluted blood is described. The approach is based on preferential electrochemical measurement of surface-bound enzyme-labeled reporter antibody (E-Ab), relative to an excess of this reagent in the sample solution. NEEIAs are carried out on microporous membranes coated with a thin, circular area of gold. The gold serves simultaneously as a working electrode and solid phase for immobilized capture anti-protein antibodies. In the assay, analyte protein is incubated concurrently with the Ab-coated gold surface and excess E-Ab conjugate. Detection of bound E-Ab is achieved by introducing the substrate for the enzyme through the back side of the membrane. The product of bound E-Ab is detected immediately by oxidation or reduction at the gold electrode, and the resulting current is proportional to the concentration of protein in the sample. The feasibility of the NEEIA approach is demonstrated via the detection of prostate-specific antigen in undiluted plasma samples (n = 64), with alkaline phosphatase as the label. Use of multiple gold films deposited on the same porous membrane to perform simultaneous NEEIAs is also described.