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Due to their unique properties, paper-based biosensors have attracted attention as inexpensive devices for on-site analysis. To achieve fast and sensitive detection of analytes, immobilization of enzymes with high apparent activities on paper is highly desirable; however, this is challenging. Herein, we report an improved approach to attach a malathion degrading enzyme, PoOPH M9 , on paper via an interlocking network of Pluronic F127 (PF127)-poly(acrylic acid)-enzyme conjugates. The addition of PF127 improved retention of enzymatic activity as the apparent kinetic constant V max of the immobilized enzyme increased two-fold compared with the paper prepared without PF127. The PF127-poly(acrylic acid)-PoOPH M9 papers provided rapid colorimetric detection of malathion at 0.1-50 mM. The detection was completed within 5 min using a smartphone and image analysis software. As a proof-of-concept, malathion-contaminated water, plant, and apple samples were analyzed with the papers successfully. This material is promising for on-site rapid analysis of malathion-contaminated samples., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)