Objective This study was conducted to investigate extent of spread of injecting methylene blue and nerve involvement in serratus anterior space. Method The ultrasound scans were performed with a GE(Venue 40) 9~13 MHz linear array transducer. Cadavers were placed in the prone position. Ribs were counted by ultrasound to locate the space between the 4th and 5th rib in the midaxillary line, and from there to the serratus anterior muscle. Under real-time ultrasound guidance, a needle was inserted within the serratus plane between the 4th and 5th rib in the midaxillary line by the Blanco method for administration of 0.4 ml/kg methylene blue. There were a total of 8 injections. After injection of 10 min, layers were separated into the anterior serratus muscle space. The extent of dye spread was measured in the serratus space, and the nerves stained were documented. Results Afterpectoralis major and minor muscles were removed respectively, structuresstained by the dye within serratus space wereidentified, including lateral cutaneous branches of 2~6 intercostal nerves, long thoracic nerve and thoracodorsal nerve stained. Spread of dye within eight serratus planes was identified by expressed in medians(P25, P75). They are 11.35(10.45,12.15)cm in the anterior axillary line,12.6(12.12,13.15)cm in the midaxillary line, 11.1(10.70,12.05)cm in the posterior axillary line, and 9.8(9.12,10.65) in the nipple line. Cephalad spread of contrast was noted in 2nd and 3rd intercostal space (75% and 25%) at anterior axillary line,(87.5% and 12.5%) at the midaxillary line, and (87.5% and 12.5%)at the anterior axillary line respectively. Conclusion This study describes that the injection spread in serratus space can be determined with the help to provide analgesia to the anterolateral chest wall.