Galectins, or beta-galactoside binding lectins, are detected deep in tumor tissue and are recognized as diagnostic and prognostic markers of cancer and other serious diseases. There is a need to develop a faster, easier, and simpler method for detecting galectins. We have succeeded in forming a mixed self-assembled monolayer (SAM) interface consisting of beta-galactoside terminated alkanethiol (lactoside protuberant dodecanethiol) and tri(ethylene glycol) (TEG) terminated short alkanethiol, which proved to be a superior protein resistant material, to enable us to develop a label-free, one-step, and highly sensitive system for detecting the expected biomarker, galectin. We successfully detected nanomolar level (~ 1 nM) galectin-4 and -8 on a 4% lactoside protrusive surface, even though the affinity between the galectins and lactoside was very weak (KD = 1 x 10(-3)~1 x 10(-6)). The combination of the suppression of background noise by filling with TEG terminated short alkanethiol and control of the ligand ratio in the interface contributed to the highly sensitive detection of galectin. We also detected galectin-4 at subten nanomolar levels even in a solution containing much higher concentrations of serum proteins (1800 times larger than the galectin concentration) without using molecule labeling or an immunological method.