The engineering of hapten-specific antibodies with affinity constant higher ( K a values > 10 10 M −1) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC ( K a1.3 × 10 10 M −1) were incubated with the “cleavable biotin”-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen–antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio > 90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.