Previous studies have shown that significant K isotope fractionation is found in continental weathering, oceanic crust subduction, and plant growth, suggesting that K isotopes could be a potentially important tracer. However, the complicated separation method and analytical conditions limit the precise analysis of potassium isotopes via MC-ICP-MS under “cold” plasma conditions. Here, an improved separation method (single column) permits the complete purification of K from the matrix elements. This method is efficient and convenient and achieves a high yield (99.5 ± 0.6%) and low blank (<10 ng K) under complex matrix extraction conditions. The high-resolution mode with a desolvating nebulizer system was used to increase the signal intensity of ⁴¹K⁺ and ³⁹K⁺ and reduce ³⁸ArH⁺, ⁴⁰ArH⁺, and some oxide/nitride interference. To eliminate scattered ions or secondary electrons, we used a new dummy bucket to collect high intensity ⁴⁰Ar⁺ beams. Besides, potassium isotopes for the standard SRM 3141a and new geological, environmental and biological reference materials such as DNC-1a, JMS-2, and GSB-14 were determined on a Nu Plasma 3 MC-ICP-MS instrument. Overall, the reproducibility of K isotope analysis was better than ±0.06‰ (2SD) that reported in most laboratories around the world. The measured δ⁴¹K values of the rock samples are homogeneous, consistent with the previous published data. However, the K isotope composition in soils, sediments, and plants is inhomogeneous and varies greatly from −0.85‰ to −0.25‰. These results show that potassium isotopes have great application potential in the geological, environmental, and biological fields.