Ultraviolet-visible spectrophotometric analysis for nucleic acids and proteins is a widespread technique. For a quick and easy use and reduce the consumption of valuable samples, micro-volume spectrophotometer is developed. In the paper, the ultraviolet-visible spectrophotometer is newly presented based on the slope measurement technique, which allows the one sequence of detection to measure the concentration of micro-volume nucleic acids or proteins samples without blank measurement. Through the experiments, we proved that the concentration can be measured without reference measurement for micro-volume samples. Additionally, in order to determine the concentration of samples, only the optical path length change through the motor drive is required, therefore there is no need to know the absolute optical path length during measurement. Furthermore, we verified that our system can even measure higher concentration sample compared to commercialized tools.