A bioassay platform using T4 bacteriophage (T4) as the specific receptor and surface plasmon resonance (SPR) as the transduction technique has been developed for the detection of Escherichia coli K12 bacteria. The T4 phages have been covalently immobilized onto gold surfaces using a self-assembled monolayer of dithiobis(succinimidyl propionate) (DTSP). Substrates of BSA/EA-T4/DTSP/Au prepared using different T4 phage concentrations have been characterized using scanning electron microscopy (SEM). The studies reveal that the use of DTSP results in a uniform binding of T4 phages onto the surface. The SPR analysis demonstrates that these BSA/EA-T4/DTSP/Au interfaces can detect the E. coli K12 with high specificity against non-host E. coli NP10 and NP30. Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 10(11) pfu ml(-1) concentration of T4 phages was used for immobilization. The surface of these chemically anchored phage substrates can be regenerated for repeated detection of E. coli K12 and can be used for detection in 7 × 10(2) to 7 × 10(8) cfu ml(-1) range. The results of these studies have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.