Biofunctional surface patterns capable of resisting nonspecific bioadsorption while retaining bioactivity play crucial roles in the advancement of life science and biomedical technologies. The currently available functional surface coatings suffer from a high level of nonspecific surface adsorption of proteins under biologically challenging conditions, leading to a loss of activity in functional moieties over time. In this study, the recently discovered facile method of temperature-induced polyelectrolyte (TIP) grafting has been used to graft two biofunctional variants (biotin and nitrilotriacetic acid, NTA) of poly(l-lysine)-grafted PEG (PLL-g-PEG) onto a titanium surface. A significant increase in the polymer adsorption was observed from the TIP-grafted surfaces assembled at 80 °C, compared to the polymer surfaces assembled at ambient temperature (20 °C). These functional PLL-g-PEG surfaces were subsequently incubated in whole human blood continuously for up to 7 days, and the TIP-grafted surfaces achieved close-to-zero nonspecific protein adsorption, as confirmed by ultrasensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). To test the maintenance of the bioactivity of the biotin and NTA moieties, submicrometer-scale mono- (biotin) and bi- (biotin/NTA) functional surface chemical patterns were fabricated via two-step TIP grafting using colloidal lithography (CL), preincubated in blood for up to 7 days and sequentially exposed to streptavidin and Ni(2+)-histidine-tagged calmodulin. The fluorescence microscopy studies revealed that the PLL-g-PEG-NTA and -biotin surfaces grafted from the TIP method were still capable of recognizing the corresponding affinity proteins for up to 1 and 7 days of preincubation in blood, respectively. These results highlight the bioresistant robustness realized by the facile TIP grafting method, which in turn preserves the activities of biofunctional moieties over a prolonged period in whole blood.