Despite the extensive development of aptamers in basic research, only a limited number have successfully progressed to clinical trials. This limitation is primarily attributed to the inherent instability of aptamers' conformation, resulting in low affinity, poor serum stability, and inconsistent potency, posing a significant challenge to their stabilization. Herein, we established a feasible strategy to develop staple aptamers using the predicted binding conformations and titration cross-linking (TTC) method. Through this strategy, we successfully synthesized various stapled sclerostin aptamers with over 70% yield. Importantly, we demonstrated that stapled aptamers significantly enhanced their affinity (approximately 20-fold) and serum stability (negligible degradation within 32 h). Moreover, in an osteogenesis imperfecta mouse model (Col1a2+/G610C mice), the stapled aptamer (named c-0127OA) exhibited a potent antagonistic effect on sclerostin, leading to enhanced anabolic bone anabolic potential. Collectively, our established stapling strategy is effective in stabilizing aptamers' conformation, with c-0127OA emerging as a promising therapeutic candidate for osteogenesis imperfecta.