A novel design of solid phase microextraction fiber containing carbon nanotube reinforced sol-gel which was protected by polypropylene hollow fiber (HF-SPME) was developed for pre-concentration and determination of BTEX in environmental waste water and human hair samples. The method validation was included and satisfying results with high pre-concentration factors were obtained. In the present study orthogonal array experimental design (OAD) procedure with OA(16) (4(4)) matrix was applied to study the effect of four factors influencing the HF-SPME method efficiency: stirring speed, volume of adsorption organic solvent, extraction and desorption time of the sample solution, by which the effect of each factor was estimated using individual contributions as response functions in the screening process. Analysis of variance (ANOVA) was employed for estimating the main significant factors and their percentage contributions in extraction. Calibration curves were plotted using ten spiking levels of BTEX in the concentration ranges of 0.02-30,000ng/mL with correlation coefficients (r) 0.989-0.9991 for analytes. Under the optimized extraction conditions, the method showed good linearity (0.3-20,000ng/L), repeatability, low limits of detections (0.49-0.7ng/L) and excellent pre-concentration factors (185-1872). The best conditions which were estimated then applied for the analysis of BTEX compounds in the real samples.