The aim of the present study was developing a surface plasmon resonance (SPR) nanosensor to detect Escherichia coli (E. coli) for the diagnosis of urinary tract infections by using surface imprinted Au nanoparticles (AuNPs) as a recognition element. In order to realize imprinting, Cu(II) ions were used to provide interaction between E. coli cell wall and amine functionalized AuNPs forming cavities on the surface of nanosensor. E. coli surface imprinted AuNPs nanosensor was characterized by using ellipsometry, contact angle measurement, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The real time detection of E. coli was evaluated by using E. coli suspensions in the concentration range of 1 × 103-0.5 × 101 CFU/mL. Combination of the signal enhancing properties of AuNPs and surface imprinting technique provided ultrasensitive detection with a comparatively low limit of detection value (1 CFU/mL) to the SPR nanosensor system. Selectivity experiments were performed by using Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. The highest response was recorded for E. coli, as expected. Additionally, the recognition of E. coli even in a complex medium such as artificial urine sample was achieved by the developed nanosensing system. Also, this chip can be used repeatedly without seen signal reducing for four-time consecutive. In the view of these results, it was emphasized that this novel sensing system has a potency for the selective, very sensitive, rapid and real time detection of causative agent in order to diagnose E. coli caused infections.