Calcium phosphate crystals, as the main component of dentin and enamel, have been widely used for the occlusion of dentinal tubules. However, the low bond strength and poor sealing effect limit their clinical practicality. In this study, a collagen/calcium dual-affinitive peptide E8DS (EEEEEEEEDSpESpSpEEDR) and nano-hydroxyapatite (nano-HAp) flocculi were developed to seal dentin tubules for reducing dentin hypersensitivity, whereas the E8DS peptides were pre-applied to improve the adhesion of occlusive hydroxyapatite coating on dentin collagen matrix for the long-lasting sealing effect and relief from hypersensitivity. Our study showed that E8DS peptides had a strong affinity with dentin collageneous matrix that almost 43.7% of initial E8DS peptides immobilized on exposed dentin samples remained detained after continuous washing by distilled water for four weeks at a rate of 1mL/min. Nano-HAp flocculi were obtained by re-neutralization of HAp-HCl solution and then brushed onto the surfaces of pre-treated human dentin disks with E8DS peptides, which showed a perfect occlusion of exposed dentinal tubules, as compared with Nano-HAp only and a commercial desensitizer, Green Or. With only around 10-min E8DS peptide pre-treatment, the occlusive mineral layers remained intact against consecutively stirred washing in phosphate-buffered saline or coke for 15min, and 6min of tooth-brushing, which implied that our E8DS peptide could comparatively improve the durability of sealant-dentin interface bonds for long-lasting dentine desensitization.