1,4-Diaminoanthraquinone (DA) as chromogen was reacted with isophorone diisocyanate (IPDI) to form a purple prepolymer, followed by interfacially polymerized with polyethylene glycol 600 to prepare amino-anthraquinone polyurethane microspheres (DAPM). The effects of emulsifier type, emulsifier dosage, emulsifying speed and chromogen dosage on the particle size of microspheres were investigated. The results showed that sodium dodecylbenzenesulphonate (SDBS) emulsifier was more suitable for the preparation of polyurethane microspheres than octaphenyl polyoxyethyiene-10 (OP-10), polyoxyethylene sorbitan monooleate (Tween80) and sorbitan monooleate (Span80). When SDBS mass fraction was 3.5% (based on the oil phase), the particle sizes of the microspheres were all around 350 nm with the narrowest particle size distribution. The average particle size decreased with the increase of emulsifying speed, when the emulsifying speed was 8000 r/min, the particle size distribution was the most concentrated. When the chromogen dosage was 1 mmol, the prepared microspheres exhibited the narrowest distribution with a particle size of 500 nm. SEM and TEM observations revealed that the as-prepared amino-anthraquinone polyurethane microspheres had a particle size of about 500 nm with smooth spherical surface and hollow internal structure. The fabric printed with DAPM had excellent color properties and color fastness.