Co‐sputtered Ni/Cu nanoalloy embedded in carbon film electrodes were applied to an isocratic high performance liquid chromatography (HPLC) electrochemical detector to analyze urinary sugar probes used to assess gastrointestinal permeability. Electrode activity was easily controlled by varying the amounts of embedded nanoalloys without changing the Ni/Cu ratio. The high electrocatalytic activity of a nanoalloy makes it possible to achieve a limit of detection (LOD) of several tens of nM, which is one or two orders of magnitude lower than that of conventional pulsed amperometric detection using a gold electrode (Au‐PAD). Moreover, Ni/Cu nanoalloy embedded in carbon film exhibits higher resistivity to electrode fouling by artificial urine components than Au‐PAD, indicating that our electrode is a promising material for detecting sugar probes in real urine samples even under normal amperometric conditions.