High-resolution analysis of stable carbon and oxygen isotopic composition (δ13C and δ18O) of tree-ring α-cellulose has been increasingly in demand for the study of climate seasonality, droughts, and tropical cyclone (hurricane) activity. The peeling–grinding method commonly used for tree-ring α-cellulose preparation is both time-consuming and labor-intensive, and often results in cross-tracheid sampling that masks high-frequency intra-annual climate signals and short-term climate events. Here, we present a new method by extracting tree-ring α-cellulose directly from wholewood spline that omits the peeling–grinding step, and allows for micro-scale δ13C and δ18O analysis that facilitates high-resolution seasonal climate study. This ultrasound-assisted non-destructive extraction method has significantly increased the output of tree-ring α-cellulose preparation while retaining the wood cell fabric. Wood cell-wall fibers collected from the α-cellulose spline were directly loaded into pre-weighed silver (or tin) capsules and packed, after oven drying, for stable oxygen (or carbon) isotope analysis. A 3.045-mm annual ring subdivided over 26 sections for δ13C and δ18O analysis revealed remarkable intra-annual isotopic variations (1.62‰ for δ13C, and 5.85‰ for δ18O). The intra-annual δ13C values inversely covary with the seasonal precipitation pattern, suggesting moisture regulation on tree-ring δ13C variations. The high-resolution δ18O analysis unravels tropical cyclone activities (Hurricanes Frances and Ivan in 2004) that are characterized by an abrupt decline in δ18O values by 2.6‰ with a “V-shape” pattern in tree-ring latewood. An algorithm is developed for establishing regional patterns of tree-ring seasonal δ13C and δ18O for the study of the mean state of climate seasonality and its anomalies.