Paper spray ionization has been demonstrated to be the most promising substrate-based source, but this technique suffers from the low desorption efficiency of target compounds and poor portability. In the current study, we describe a portable paper-based electrospray ionization (PPESI) in which a piece of triangle paper and adsorbent are packed sequentially into a modified disposable micropipette tip. This source not only captures the feature of paper spray and adsorbent for highly efficient suppression of sample matrixes for target compound analysis but also takes advantage of a micropipette tip to prevent spray solvent from rapid evaporation. The performance of developed PPESI depends on the type and amount of packed adsorbent, paper substrate, and spray solvent and applied voltage. Moreover, by contrast to other related sources, the analytical sensitivity and the spray duration of PPESI in tandem with MS have been improved by factors of 2.8-32.3 and 2.0-13.3, respectively. Based on its high accuracy (>96%) and precision (less than 3% relative standard deviation), the PPESI coupled to a mass spectrometer has been used to determine diverse therapeutic drugs and pesticides in complex biological (e.g., whole blood, serum, and urine) and food (e.g., milk and orange juice) matrixes, and the limits of detection and quantification were 2-4 pg mL-1 and 7-13 pg mL-1, respectively. Taking the portability, high sensitivity, and repeatability, the technique may be a promising alternative for complex sample analysis.