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DNA nanomachines with artificial intelligence have attracted great interest, which may open a new era of precision medicine. However, their in vivo behavior, including early diagnosis and therapeutic effect are limited by their targeting efficiency. Here, a tetrahedral DNA framework (TDF)‐based nanodevice for in vivo near‐infrared (NIR) diagnosis of early‐stage AKI is developed. This nanodevice comprises three functional modules: a size‐tunable TDF nanostructure as kidney‐targeting vehicle, a binding module for the biomarker kidney injury molecule‐1 (Kim‐1), and a NIR signaling module. The cooperation of these modules allows the nanodevice to be selectively accumulated in injured kidney tissues with high Kim‐1 level, generating strong NIR fluorescence; whereas the nanodevice with the proper size can be rapidly cleared in healthy kidneys to minimize the background. By using this nanodevice, the early diagnosis of AKI onset is demonstrated at least 6 h ahead of Kim‐1 urinalysis, or 12 h ahead of blood detection. It is envisioned that this TDF‐based nanodevice may have implications for the early diagnosis of AKI and other kidney diseases. [ABSTRACT FROM AUTHOR]