Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.