Background: Ubiquitylation plays an important role in regulating protein functions. Recently, experimental methods were developed toward effective identification of ubiquitylation sites. To efficiently explore more undiscovered ubiquitylation sites, this study aims to develop an accurate sequence-based prediction method to identify promising ubiquitylation sites. Results: We established an ubiquitylation dataset consisting of 157 ubiquitylation sites and 3676 putative non-ubiquitylation sites extracted from 105 proteins in the UbiProt database. This study first evaluates promising sequence-based features and classifiers for the prediction of ubiquitylation sites by assessing three kinds of features (amino acid identity, evolutionary information, and physicochemical property) and three classifiers (support vector machine, k-nearest neighbor, and NaïveBayes). Results show that the set of used 531 physicochemical properties and support vector machine (SVM) are the best kind of features and classifier respectively that their combination has a prediction accuracy of 72.19% using leave-one-out cross-validation. Consequently, an informative physicochemical property mining algorithm (IPMA) is proposed to select an informative subset of 531 physicochemical properties. A prediction system UbiPred was implemented by using an SVM with the feature set of 31 informative physicochemical properties selected by IPMA, which can improve the accuracy from 72.19% to 84.44%. To further analyze the informative physicochemical properties, a decision tree method C5.0 was used to acquire if-then rule-based knowledge of predicting ubiquitylation sites. UbiPred can screen promising ubiquitylation sites from putative non-ubiquitylation sites using prediction scores. By applying UbiPred, 23 promising ubiquitylation sites were identified from an independent dataset of 3424 putative non-ubiquitylation sites, which were also validated by using the obtained prediction rules. Conclusion: We have proposed an algorithm IPMA for mining informative physicochemical properties from protein sequences to build an SVM-based prediction system UbiPred. UbiPred can predict ubiquitylation sites accompanied with a prediction score each to help biologists in identifying promising sites for experimental verification. UbiPred has been implemented as a web server and is available at http://iclab.life.nctu.edu.tw/ubipred.