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Uric acid and creatinine are essential biomarkers for many diseases, such as gout, hyperuricemia, kidney diseases and heart diseases. Electrochemical biosensors are promising candidates for detecting uric acid and creatinine. However, the sensors always suffer from low stability that would limit their practical applications. In this work, we report a new multilayer enzyme matrix to enhance the room-temperature storage stabilities of uric acid and creatinine biosensors significantly. The enzymes are first dropped on the electrode surface directly, then a layer of glutaraldehyde was deposited on the surface of the enzyme layer, and after that, another layer of a polyvinyl alcohol (PVA)/poly(ethylene glycol) (PEG) composite was further placed on the surface of the glutaraldehyde layer, with drying in between. The stabilities of uric acid and creatinine biosensors were enhanced significantly, and the sensors can maintain highly stable sensing performance for over 4 months with a storage at room temperature. It is anticipated that this work could open new avenues for the practical applications of the uric acid and creatinine biosensors. [Display omitted] • The work reports a new multilayer enzyme matrix to enhance the room-temperature storage stabilities of biosensors. • The method is based on a sequential deposition of enzyme, glutaraldehyde, and polyvinyl alcohol/poly(ethylene glycol). • The sensors can maintain highly stable sensing performance for over 4 months with a storage at room temperature. • The sensors can perform accurate detection of uric acid and creatinine in whole blood samples. [ABSTRACT FROM AUTHOR]