In this work, mixed polymer brushes coating based on poly(2-methyl-2-oxazoline) (PMOXA)/poly(4-vinyl pyridine) (P4VP) was prepared by simultaneously grafting amine-terminated PMOXA and thiol-terminated P4VP onto poly(dopamine) (PDA)-modified substrates. The coatings were characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, zeta potential measurements, and the static water contact angle (WCA) tests. The results demonstrated that the coating based mixed PMOXA/P4VP brushes with desired surface composition could be obtained by simply maintaining their percentage in the mixture of PMOXA and P4VP solutions. Moreover, the zeta potential and the WCA of mixed brushes modified surfaces could be tuned by changing the environmental pH value and surface compositions. Finally, the switchable behavior of PMOXA/P4VP based coatings toward pepsin adsorption was investigated by fluorescein isothiocyanate-labelled pepsin assay and surface plasmon resonance. The results showed that by adjusting the fraction of PMOXA or P4VP, the PMOXA/P4VP mixed brushes coated surfaces could adsorb large amounts of pepsin at pH 3, but more than 92% of the adsorbed protein could be desorbed at pH 7.