This study presents a paper-based thin-layer optical cuvette for absorption spectroscopy with a pneumatically driven pump for the introduction of an aqueous sample. The three major components, a sample reservoir, an open-channel as an optical path and a pneumatic pump, are patterned in polypropylene paper using an electronic cutting machine. When sealed with transparent tape, the patterned paper serves as the side walls of the paper-based cuvette with an integrated pneumatic pump. Due to the reduced pressure inside the open-channel caused by the depression of the pump with a finger, aqueous samples spotted onto the reservoir are introduced into the open-channel of the paper-based cuvette. We demonstrated that the paper-based cuvettes are compatible with conventional spectrophotometers for absorption spectroscopic measurements and capable of quantifying the concentrations of various colored dyes in aqueous samples.