An enrichment and determination method based on liquid chromatographically analysis and cloud point extraction (CPE) has been developed for trace levels of B vitamins (B1, B9 and B12) in the proposed study. Vitamin molecules were drawn into the non-ionic surfactant phase of Polyethylene Glycol (PEG-6000) in the presence of pH 9.0 medium. The surfactant-rich phase separated by centrifugation and then dissolved with 700 µL of ethanol. The obtained ethanol phase was filtered by 0.45-micron filter prior to the HPLC analysis. All parameters affecting the CPE method such as pH, buffer volume, incubation time, surfactant and electrolyte concentration, solvent for the surfactant-rich phase and its amount have been individually studied and optimized step by step. After the optimization of all parameters of the CPE process, the detection limits of the developed method for B1, B9 and B12 vitamins were calculated as 1.42 ng mL-1, 7.14 ng mL-1 and 14.28 ng mL-1 , respectively. The linear working ranges for three vitamin molecules was obtained in the range of 5.0-500.0 ng mL-1. After CPE procedure, determination of vitamin molecules was carried out by using HPLC system with diode array detector(DAD) at 244 nm for vitamin B1, 285 nm for vitamin B9, and 361 nm for vitamin B12, respectively