Heavy metal pollution is one of the most serous environmental issues globally. To evaluate the metal pollution in Jiangsu Province of China, the total concentrations of heavy metals in sediments and fishes from the Yangtze River and Taihu Lake were analyzed. Ecological risk of sediments and human health risk of fish consumption were assessed respectively. Furthermore, toxicity of samples on expression of the stress responsive genes was evaluated using microbial live cell-array method. The results showed that the heavy metals concentrations in sediments from the Yangtze River were much higher than those in sediments from the Taihu Lake. However, the fishes from the Taihu Lake had higher concentrations of heavy metals than fishes from the Yangtze River. Ecological risk evaluation showed that the heavy metal contaminants in sediments from the Yangtze River posed higher risk of adverse ecological effects, while sediments from the study areas of Taihu Lake were relatively safe. Health risk assessment suggested that the heavy metals in fishes of both Yangtze River and Taihu Lake might have risk of adverse health effects to human. The toxicity assessment indicated that the heavy metals in these sediments and fishes showed transcriptional effects on the selected 21 stress responsive genes, which were involved in the pathways of DNA damage response, chemical stress, and perturbations of electron transport. Together, this field investigation combined with chemical analysis, risk assessment and toxicity bioassay would provide useful information on the heavy metal pollution in Jiangsu Province.