Biological toxicity testing plays an essential role in identifying the possible negative effects induced by substances such as organic pollutants or heavy metals. As an alternative to conventional methods of toxicity detection, paper-based analytical device (PAD) offers advantages in terms of convenience, quick results, environmental friendliness, and cost-effectiveness. However, detecting the toxicity of both organic pollutants and heavy metals is challenging for a PAD. Here, we show the evaluation of biotoxicity testing for chlorophenols (pentachlorophenol, 2,4-dichlorophenol, and 4-chlorophenol) and heavy metals (Cu2+, Zn2+, and Pb2+) by a resazurin-integrated PAD. The results were achieved by observing the colourimetric response of bacteria (Enterococcus faecalisandEscherichia coli) to resazurin reduction on the PAD. The toxicity responses ofE. faecalis-PAD andE. coli-PAD to chlorophenols and heavy metals can be read within 10 min and 40 min, respectively. Compared to the traditional growth inhibition experiments for toxicity measuring which takes at least 3 h, the resazurin-integrated PAD can recognize toxicity differences between studied chlorophenols and between studied heavy metals within 40 min.