Microplastics (MPs) are universally present in the ecosystem and pose great threats to the environment and living organisms. Research studies have shown that small MPs (<50 μm in diameter) are especially toxic and account for more than half of all MPs collected in the Atlantic Ocean. Nevertheless, current methods for the detection and analysis of MPs are incapable of achieving rapid and in situ analysis of small MPs in the biota to ultimately enable the study of their biological effects. In this work, we report a method that allows rapid in situ identification and spatial mapping of small MPs directly from paramecia with high accuracy by acquiring chemical composition information using secondary-ion mass spectrometry (SIMS) imaging. Specifically, six types of common MPs (polymethyl methacrylate, polyvinyl chloride, polypropylene, polyethylene terephthalate, polyglycidyl methacrylate, and polyamide 6) with a diameter of 1-50 μm were simultaneously imaged with high chemical specificity at a spatial resolution of 700 nm. In situ spatial mapping of a group of MPs ingested by paramecia was performed using SIMS fragments specific to the plastic composition with no sample pretreatment, revealing the aggregation of MPs in paramecia after ingestion. Compared with existing methods, one additional advantage of the developed method is that the MPs and the organism can be analyzed in the same experimental workflow to record their fingerprint spectra, acquiring biochemical information to evaluate MP fate, toxicity, and the MP-biota interaction.