Recent thrilling progress in large-scale text-to-image (T2I) models has unlocked unprecedented synthesis quality of AI-generated content (AIGC) including image generation, 3D and video composition. Further, personalized techniques enable appealing customized production of a novel concept given only several images as reference. However, an intriguing problem persists: Is it possible to capture \textbf{multiple, novel concepts} from \textbf{one single reference image}? In this paper, we identify that existing approaches fail to preserve visual consistency with the reference image and eliminate cross-influence from concepts. To alleviate this, we propose an attention calibration mechanism to improve the concept-level understanding of the T2I model. Specifically, we first introduce new learnable modifiers bound with classes to capture attributes of multiple concepts. Then, the classes are separated and strengthened following the activation of the cross-attention operation, ensuring comprehensive and self-contained concepts. Additionally, we suppress the attention activation of different classes to mitigate mutual influence among concepts. Together, our proposed method, dubbed \textbf{DisenDiff}, can learn disentangled multiple concepts from one single image and produce novel customized images with learned concepts. We demonstrate that our method outperforms the current state of the art in both qualitative and quantitative evaluations. More importantly, our proposed techniques are compatible with LoRA and inpainting pipelines, enabling more interactive experiences.