The preS1 domain present on hepatitis B and D virus particles is an intrinsically disordered peptide (IDP) essential for recognizing the host receptor, sodium taurocholate co-transporting polypeptide (NTCP). However, it remains poorly understood how the disordered preS1 peptide achieves high-affinity binding through its conformational change. Here, we reveal a stepwise binding process through which multiple regions within preS1 cooperate to establish the stable binding to NTCP. By combining structure-based molecular simulations with virological assays, we show that Asn9^preS1, Gly12^preS1, and His17^preS1 primarily mediate the NTCP binding by forming a tandem loop core structure that docks into the bile acid tunnel of NTCP, creating an extensive binding interface. Another interaction via Trp41^preS1 stabilizes the flexible preS1 33–48 aa stretch embedded onto the NTCP outerface, particularly involving Asn87^NTCP and Tyr146^NTCP. Mutation in these key residues abolishes virion infectivity in cell culture and mouse models, demonstrating the biological significance of this binding maturation. These findings propose a multistep mechanism for viral IDP-mediated receptor recognition that ensures high virion infectivity and strict host specificity.