Abstract Cisplatin, an anti-cancer drug, has been used extensively and has become a “gold standard” treatment for various types of cancer. However, overdosage has serious side effects. Thus, this drug should not be over-administered. The current study presents a label-free but sensitive fluorescent sensor for cisplatin detection based on G-quadruplex (GQ) and fluorescent dye Thioflavin T (ThT). In the absence of cisplatin, ThT binds to GQ, resulting in high fluorescence emission. In the presence of cisplatin, the GQ structure is destroyed after cisplatin–DNA adduct formation, leaving no binding scaffold for ThT, thereby causing a decrease in ThT fluorescence. The amount of cisplatin can be quantified from the changes in ThT fluorescence intensity. Parameters that affect the sensor performance were tested, including pH and concentration of potassium phosphate buffer, type of GQ, number of quartets, loop length, incubation time, and temperature. The sensor provided a linear dynamic range of 10–500 nM with limit of quantification at 10 nM. The sensor was successfully applied to detect cisplatin in urine samples from patients undergoing treatment with high accuracy (recoveries between 74 ± 14 and 112.8 ± 5.0%) and precision (relative standard deviations between 1.15% and 2.12%).