Doxorubicin (DOX) is a potent chemotherapeutic agent known to induce cardiotoxicity. Here we applied one-dimensional scanning multiphoton imaging to investigate the derangement of cardiac dynamics induced by DOX on a zebrafish model. DOX changed the cell morphology and significantly prolonged calcium transient and sarcomere contraction, leading to an arrhythmia-like contractile disorder. The restoration phase of calcium transient dominated the overall prolongation, indicating that DOX perturbed primarily the protein functions responsible for recycling cytosolic calcium ions. This novel finding supplements the existing mechanism of DOX cardiotoxicity. We anticipate that this approach should help mechanistic studies of drug-induced cardiotoxicity or heart diseases.