High-quality labeled data are crucial prerequisites for ensuring the effectiveness of fault diagnosis methods based on deep learning technology. However, in practical scenarios, providing abundant training data with accurate labels for these approaches is unfeasible owing to the constraints imposed by the operating and working conditions. To tackle this realistic challenge, we propose an innovative feature separation simulation-assisted transfer framework (FSSATF) for the fault diagnosis of rotating machinery. The primary concept of FSSATF is to leverage dynamic simulation-assisted data as a surrogate for the labeled data of actual equipment and integrate the feature separation network to explicitly extract domain-independent and fault-discriminative features from the simulated and actual domains, facilitating knowledge transfer and enhancing fault diagnosis capabilities. Specifically, we design a feature separation network consisting of two feature extractors. The special feature extractor is trained with the proposed target domain classification loss to explicitly separate the noisy features from the actual data. Moreover, our proposed domain adaptive loss function effectively narrows the distribution discrepancy between the simulated and actual data, promoting the shared feature extractor to capture domain-invariant and fault-discriminative features. Additionally, clustering learning is embedded into FSSATF to minimize the distance between samples of the same category, strengthening the model's capabilities for feature extraction, and improving its performance in real machinery fault diagnosis. Artificially damaged and run-to-failure datasets were employed to validate the effectiveness and superiority of FSSATF. The comparative analysis results demonstrate that the fault diagnosis performance surpasses those of other advanced transfer learning fault diagnosis methods.