A micro-patterned Prussian Blue (PB) array of nanometer thickness was fabricated using soft photolithography and voltammetric deposition for electrocatalytic detection of biomolecules. A self-assembled alkylthiol monolayer on gold was photooxidized by UV irradiation with a TEM mask, generating an imprinted pattern into which a PB microarray was subsequently deposited. Cyclic voltammetric and microscopic characterization indicates that the PB arrays demonstrate improved electrochemical stability as compared to conventional PB films, particularly at neutral pH, allowing scores of potential cyclings with little film deterioration. The PB film thickness in the array can be conveniently controlled by varying deposition time. Facile electron transfer was observed on the array electrode and the effect of the SAM layer on electrochemical response was investigated. The use of PB arrays for electrocatalytic analysis of biologically significant molecules was demonstrated with ascorbic acid (AA). The calibration curve for AA shows a linear relationship over the concentration range of 1.0 x 10(-5) to 8 x 10(-7) M with a correlation coefficient of 0.99. The detection limit was determined to be 1 x 10(-8) M, which is about 100 times lower than the reported value on a conventional PB electrode. The method developed here provides a new functional surface for bioanalysis using electrocatalytic principles.