A Prussian blue analogue with cobalt (Na2‐xCo[Fe(CN)6]1‐y, PB‐Co), which is often used as sodium‐ion battery material, was found to be an excellent sensing material toward some biomolecules by calcination. PB‐Co calcined at 750 °C for 1 h (PB‐Co750‐1) has the improved defects to facilitate the adsorption and the catalytic oxidation of ascorbic acid (AA), dopamine (DA) and uric acid (UA). PB‐Co750‐1 modified on screen printed carbon electrode (SPE) for the ultrasensitively simultaneous determination of AA, DA and UA demonstrates the wide linear range, the low limit of detection (LOD) and the good reproducibility. In addition, the redox of the analytes was confirmed as the diffusion‐controlled process via the kinetic study. The recoveries of AA, DA and UA in real samples were found between 91 % to 114 %. AA and UA in tablets, serums and urines could be determined by PB‐Co750‐1/SPE with the satisfactory results.