A bio-metal-organic framework (bio-MOF) derived from the amino acid L-serine has been prepared in bulk form and evaluated as sorbent for the molecular recognition and extraction of B-vitamins. The functional pores of bio-MOF exhibit high amounts of hydroxyl groups jointly directing other supramolecular host-guest interactions thus providing the recognition of B-vitamins in fruit juices and energy drinks. Single-crystal X-ray diffraction studies reveal the specific B-vitamin binding sites and the existence of multiple hydrogen bonds between these target molecules and the framework. It offered unique snapshots to accomplish an efficient capture of these solutes in complex aqueous matrices. Four B-vitamins (thiamin, nicotinic acid, nicotinamide, and pyridoxine) were investigated. They were eluted from the sorbent with phosphate buffer at pH 7 and analyzed by HPLC with UV detection. The sorbent was compared with commercial C18 cartridges. Following the procedure, acceptable reproducibility (RSD values < 14%) was achieved, and the detection limits were in the range 0.4 to 1.4 ng mL−1. The method was applied to the analysis of energy drink and juice samples and the recoveries were between 75 and 123% in spiked beverage samples.