A distance-dependent two-way magnetic resonance tuning platform combined with dual-contrast enhanced subtraction imaging enables quantitative sensing and imaging in deep tissues with minimal background noise. Distance-dependent magnetic resonance tuning (MRET) technology enables the sensing and quantitative imaging of biological targets in vivo, with the advantage of deep tissue penetration and fewer interactions with the surroundings as compared with those of fluorescence-based Forster resonance energy transfer. However, applications of MRET technology in vivo are currently limited by the moderate contrast enhancement and stability of T-1-based MRET probes. Here we report a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T-1 and T-2 magnetic resonance signals that is coupled with dual-contrast enhanced subtraction imaging. This integrated platform achieves a substantially improved contrast enhancement with minimal background signal and can be used to quantitatively image molecular targets in tumours and to sensitively detect very small intracranial brain tumours in patient-derived xenograft models. The high tumour-to-normal tissue ratio offered by TMRET in combination with dual-contrast enhanced subtraction imaging provides new opportunities for molecular diagnostics and image-guided biomedical applications.