Crystallization of Fe–Ti oxides can induce significant Titanium (Ti) isotope fractionation during magmatic differentiation. As such, rutile (TiO₂), a common Ti-rich mineral present in igneous, metamorphic, and sedimentary rocks, can serve as a tracer of the geologic processes. A new method to determine the Ti mass-dependent isotope fractionation of rutiles with high precision and high spatial resolution was developed using femtosecond laser ablation multi-collector inductively coupled plasma mass spectrometry (fs-LA-MC-ICP-MS). A high sensitivity cone combination (Standard sampler cone + X skimmer cone) can increase the Ti signals, which permits ablation of rutiles with a low ablation frequency of 1 Hz and analysis with high spatial resolution. However, a signal smoothing device is necessary to overcome the signal fluctuation issue that commonly occurs in the modern low-dispersion ablation cell at a low ablation frequency. Our results show that changing laser parameters (spot size and laser fluence) or signal mismatch between samples and references would bias toward higher δ⁴⁹Ti values (0.16‰ to 0.59‰) in dry plasma, which can be suppressed by using wet plasma conditions instead. After these optimizations, the spatial resolution of our method at horizontal- and vertical-axis can be promoted to 10 μm and ≈4 μm, respectively, meanwhile ensuring accurate δ⁴⁹Ti results with a precision of ±0.10‰. The long-term measurements of a rutile USA75 by fs-LA-MC-ICP-MS show a good reproducibility of δ⁴⁹Ti (±0.11‰). Measurements of 9 natural rutile crystals by both solution MC-ICP-MS and fs-LA-MC-ICP-MS provide consistent δ⁴⁹Ti values, confirming the robustness of our fs-LA-MC-ICP-MS method. A significant variation of δ⁴⁹Ti (up to ≈2.94‰) among the 12 rutiles was observed, indicating that Ti isotopes in rutiles can be a useful geochemical tracer. Four rutile crystals (e.g., USA75, Bra12, Sco2 and Bra6) are shown to be homogeneous in δ⁴⁹Ti within an uncertainty of <0.13‰ and can therefore serve as reference materials for future in situ Ti isotope ratios measurement.