Abstract: A novel green synthesis strategy was proposed for preparation of multi-template molecularly imprinted biopolymers (mt-MIBP) in aqueous media with less consumption of organic solvents, which were subsequently used as sorbents of ultrasound-assisted dispersive solid-phase extraction (d-SPE) for simultaneous recognition and efficient separation of B-family vitamins in juice samples followed by high performance liquid chromatography (HPLC) determination. The obtained mt-MIBP was fully characterized by SEM, FT-IR, TEM and BET. It offered high binding capacity, good selectivity and fast dynamics toward all the templates. Involved parameters in the d-SPE efficiency such as mt-MIBP mass, sonication time, and eluting/washing solvents' types and volumes were concurrently investigated by central composite design (CCD) with rapidity and reliability. Under the optimum conditions, the developed mt-MIBP-d-SPE-HPLC method exhibited wide linear range, low limits of detection (LODs) and quantification (LOQs) within 1.2-5.5 μg L-1 and 4.0-18.4 μg L-1, respectively, and appropriate repeatability (relative standard deviation values (RSDs) below 4.2%, n=4). The high selectivity of this method makes it suitable for successful monitoring of vitamins in juice samples with satisfactory recoveries of 75.8-92.7%, 83.3-92.5% and 84.7-93.8% for vitamins riboflavin (B2), nicotinamide (B3) and pyridoxine (B6), respectively. The present study implied highly promising perspectives of water-compatible eco-friendly mt-MIBP for highly effective multi-residue analysis in complicated matrices