A novel paper spray ionization mass spectrometric(PSI-MS) method was developed for the direct detection of biofluid.The agarose hydrogel coated paper substrate was used to alleviate the lateral diffusion of polar extraction solvent to achieve both high extraction efficiency and ambient ionization,whereupon liquid-liquid extraction occurred between the biofluid sample droplet and the polar extract/spray solvent.Hence,the overall sensitivity of hydrogel coated PSI-MS could be improved greatly due to better extraction efficiency.Meanwhile,the evaporation rate for the extract/spray solvent was reduced due to its less diffusion,and a longer and more stable continuous mass spectrometry signal was obtained.In addition,ethyl acetate was used here as spray and extract solvents to form liquid-liquid extraction,high voltage DC was applied on the hydrogel coated paper substrate where sample was placed to achieve fast and accurate quantification of low abundance therapeutic drugs in biofluid samples.Compared with PSI-MS,the hydrogel coated PSI-MS extended the spray time by nearly 10 times and the signal for analyte by about 100 times.The method was applied in the quantitative detection of drugs in whole blood and urine samples with the limits of detection(LODs) decreased by 7-33 times.The hydrogel coated PSI-MS showed its great potential in the area of on site clinical diagnosis,owing to its simple preparation,high sensitivity and convenient operation.