Abstract: DNA analysis is the core of biotechnology applied in petroleum resources and engineering. Traditionally accurate determination of DNA purity and concentration by spectrometer is the first and critical step for downstream molecular biology research. In this study, three different spectrophotometry methods, BPM, NDTT and NPMTTZ were compared for their performance in determining DNA concentration and purity in 32 oil samples, and molecule methods like quantitative real-time PCR (qPCR) and high-throughput sequence were also performed to help assess the accuracy of the three methods in determining DNA concentration and purity. For ordinary heavy oil (OHO), extra heavy oil (EHO) and super heavy oil (SHO), the characteristics of high viscosity (η), density (ρ) and resin plus asphaltene content will affect the DNA extraction and UV determination. The DNA concentration was decreased as density increased: OHO (11.46 ± 18.34 ng/μL), EHO (6.68 ± 9.67 ng/μL) and SHO (6.20 ± 7.83 ng/μL), and the DNA purity was on the reverse: OHO (1.31 ± 0.27), EHO (1.54 ± 0.20), and SHO (1.83 ± 0.32). The results suggest that spectrophotometry such as BPM and NPMTTZ are qualitatively favorite methods as the quick non-consumable methods in determining DNA concentration and purity of medium oil and heavy oil.