A novel electrochemical paper-based microfluidic chip was firstly developed to simultaneously detect cadmium (Ⅱ) and lead (Ⅱ) in vegetable and fruit samples. The patterned filter paper was prepared through the printing of three-electrode patterns on filter paper using an automatic screen-printing machine. Portable and low-cost (less than $1) electrochemical paper-based chips are prepared by filling conductive ink and hot pressing. The paper-based chip could realize signal amplification through gold nanoparticles and seed solutions. Cadmium (Ⅱ) and lead (Ⅱ) were sensitively detected by their aptamers labeled with methylene blue and ferrocene, separately. Under the optimal experimental parameters, the paper-based chip detected cadmium (Ⅱ) and lead (Ⅱ) as low as 23.31 and 46.23 pmol/L (3σ) with a wide linear range from 0.1 to 1000 nmol/L and exhibited excellent selectivity. The RSD was 6.41% (cadmium (Ⅱ)) and 4.20% (lead (Ⅱ)). Compared with other methods, the paper-based chip could complete the detection within 15 min and could be stored at −20 °C for 5 days. Furthermore, the results for vegetable and fruit samples were agreed with the results of the graphite furnace atomic absorption spectrophotometer, in which the recovery rates were 93.20%–95.80%. An electrochemical paper-based chip for simultaneous detection of cadmium and lead according to changes in electrochemical signals was established. Display omitted •An ultrasensitive electrochemical paper-based microfluidic chip was developed to simultaneously detect cadmium (Ⅱ) and lead (Ⅱ) in fruit and vegetable.•The electrochemical paper-based chip was simple to make through an automatic screen-printing machine, easy to carry and low cost (less than $1).•The electrochemical paper-based chip displayed low detection limit of 46.23 pmol/L for lead (Ⅱ) and 23.31 pmol/L for cadmium (Ⅱ).