A horseradish peroxidase (HRP) biosensor was constructed using binary self-assembled monolayers (SAM) of 11-mercaptoundecanoic acid (MUA) and thiolactic acid (TLA) on gold surface. The advantages of using mixed SAM for the enzyme immobilization is that the long carbon chain molecules act as a support for the enzyme while the short chain molecules favor the electron transfer process. In order to obtain this modified surface, the gold electrode was incubated in a solution containing different proportions of MUA and TLA and the best concentration ratio of these molecules was 0.5 and 1.0 mmol L −1 , respectively. The preparation steps and the biosensor response were monitored by electrochemical techniques. The biosensor proposed was applied to determine hydroquinone in a 0.10 mol L −1 phosphate buffer solution containing H 2 O 2 0.3 mmol L −1 . The Au-SAM mix -HRP electrode, in the presence of hydrogen peroxide, catalyzes the oxidation of hydroquinone to the corresponding quinone, which is electrochemically reduced back to hydroquinone at −0.08 V vs Ag/AgCl. The analytical curve was linear for hydroquinone concentrations from 5.0 to 30 μmol L −1 and the detection limit was 1.26 μmol L −1 . The lifetime of this biosensor was 15 days. The modified electrode displayed good reproducibility, sensitivity and stability for the determination of hydroquinone.The modification of electrode surfaces using the self-assembled monolayers (SAM) technique is more convenient, because they form spontaneously, easy to handle mechanically and relatively stable in electrolyte solutions. 1 The main interest in developing mixed selfassembled monolayers (SAM mix ) is related to the development of attractive methods to promote different arrangements on the electrode surface, which enables the control of specific reaction sites. In this way, SAM mix can be configured as an appropriate platform for the immobilization of biomolecules 2 which have been frequently applied in electroanalysis for the development of biosensors, 3,4 since well-organized and compact monolayers present advantages like selectivity, sensitivity and reduced overpotentials in electrocatalytic reactions. The SAM mix can be obtained by combining the properties of alkanethiols with different carbon chain lengths or different functional group. 5 Several methods for the preparation of mixed SAMs are reported in the literature, being the most commonly used the coadsorption of different thiols, 6,7 and also by electrochemical substitution modification. 8,9 The SAM mix obtained through co-adsorption of different functional groups was described by Ngunjiri et al, 10 formed by molecules octadecanethiol and 11-mercaptoundecanoic acid for immobilization of proteins. Another example, Ji et al. 11 developed the SAM mix formed by thioctic acid (T-COOH) and thioctic acid amide (T-NH 2 ) which was used to immobilize tyrosinase to construct an electrochemical biosensor for phenolic compounds. Studies with this biosensor showed that mixed SAMs improved protein adsorption and they were more co...