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Several tropical fever viruses transmitted by mosquitoes including zika, dengue, and chikungunya , are becoming a serious problem in global public health. Simple diagnostic tools in early stages are strongly required to monitor and prevent these diseases. Paper diagnostic platforms can provide a solution for these needs, with integration of fluidic control techniques and isothermal amplification methods. Here, we demonstrate a Lab-on-paper for all-in-one molecular diagnostics of zika, dengue, and chikungunya virus from human serum. The entire process of nucleic acid testing that involves sampling, extraction, amplification, and detection is simply operated on a single paper chip. Based on the engineered structure of paper materials and dried chemicals on the all-in-one chip, serum samples containing the target virus RNA were simply added by automatic flow from distilled water injection. Target RNA molecules were concentrated on the binding pad with chitosan and then transported to reaction pads following a pH increase for specific reverse transcription loop-mediated isothermal amplification with fluorescence signal generation. Three targets, zika virus, dengue virus, and chikungunya virus, in human serum were simultaneously detected on the all-in-one paper chip within 60 min at 65 °C. The all-in-one paper chip can be used as a real-time quantitative assay for 5–5000 copies of zika virus RNA. This all-in-one device was successfully used with 5 clinical specimens of zika and dengue virus from real patients. We believe that the proposed all-in-one paper chip can provide a portable, low-cost, user-friendly, sensitive, and specific NAT platform with great potential in point-of-care diagnostics. Image 1 • All-in-one nucleic acid testing paper chip from sample to result was demonstrated. • The entire nucleic acid testing process, sampling, extraction, amplification, and detection were conducted on single device by simple injection of sample. • Zika, dengue, and chikungunya spiked in serum samples were simultaneously detected and quantified within 60 min by RT-LAMP. • Small volumes, 5 clinical samples, and 5–5000 RNA copies were analyzed in real-time. • A fully-integrated, portable, low-cost, ready-to-use, easy-to-fabricate, sensitive, and specific paper-based nucleic acid testing system was developed. [ABSTRACT FROM AUTHOR]