An efficient solid-phase method has been reported to prepare well-defined lysine defect dendrimers. Using orthogonally protected lysine residues, pure G2 to G4 lysine defect dendrimers were prepared with 48–95% yields within 13 h. Remarkably, high-purity products were collected via precipitation without further purification steps. This method was applied to prepare a pair of 4-carboxyphenylboronic acid-decorated defect dendrimers ( 16 and 17 ), which possessed the same number of boronic acids. The binding affinity of 16 , in which the ε-amines of G1 lysine are fractured, for glucose and sorbitol was 4 times that of 17 . This investigation indicated the role of allocation and distribution of peripheries for the dendrimer’s properties and activity.