Microfluidic-based portable devices for stool analysis are important for detecting established biomarkers for gastrointestinal disorders and understanding the relationship between gut microbiota imbalances and various health conditions, ranging from digestive disorders to neurodegenerative diseases. However, the challenge of processing stool samples in microfluidic devices hinders the development of a standalone platform. Here, we present the first microfluidic chip that can liquefy stool samples via acoustic streaming. With an acoustic transducer actively generating strong micro-vortex streaming, stool samples and buffers in microchannel can be homogenized at a flow rate up to 30 μL min-1. After homogenization, an array of 100 μm wide micropillars can further purify stool samples by filtering out large debris. A favorable biocompatibility was also demonstrated for our acoustofluidic-based stool liquefaction chip by examining bacteria morphology and viability. Moreover, stool samples with different consistencies were liquefied. Our acoustofluidic chip offers a miniaturized, robust, and biocompatible solution for stool sample preparation in a microfluidic environment and can be potentially integrated with stool analysis units for designing portable stool diagnostics platforms.