Atruncated aptamer (designated A24-3) was identifiedthat specifically binds to bovine pregnancy-associated glycoproteins (bPAG9) with a low dissociation constant (2.04nM) through two truncation approaches. Circular dichroism spectroscopy indicated that A24-3 formed parallel G-quadruplexes, which was subsequently confirmed using nuclear magnetic resonance (NMR) spectroscopy. Furthermore, a molecular dynamics simulation was employed to investigate the recognition mechanism of A24-3 and bPAG9. Interaction analysis showed that A24-3 folded into a parallel G-quadruplex structure with three G-tetrads, primarily through numerous hydrogen bonds and hydrophobic and π-π interactions. Finally, a novel colorimetric aptasensor was developed for detecting bPAG9 using A24-3 and an Fe-based metal-organic framework as target recognition elements and enzyme mimics, respectively. The method demonstrated a broad detection range from 0.5 to 50ng/mL, with a low detection limit of 0.03ng mL-1, and exhibited a good recovery (91.0-102%) for bPAG9-spiked serum samples. Additionally, the aptasensor was successfully applied todetecting the pregnancy-specific biomarker bPAGs in serum samples.