We have developed an electrochemical biosensor to measure the lysine concentration in blood. The lysine biosensor has a simple design and can complete measurements of a 5 μL sample in 2 min, enabling rapid measurement in clinical practice. The target molecules are catalyzed by enzymes encapsulated in carboxymethyl cellulose on the working electrodes to produce potassium ferrocyanide as an electron mediator that is detected on the electrodes amperometrically. In a clinical application, the sensor was used to measure blood lysine concentrations in 47 pediatric surgical patients. The results showed that the lysine level was correlated with the transthyretin level, a conventional nutritional indicator, confirming its usefulness for nutritional assessment. This sensor can be used for postoperative nutritional management and treatment of rare diseases such as lysinuric protein intolerance. The platform also has a versatile design for the detection of other amino acids in addition to lysine.