Heteroatom doped carbon materials have attracted intense attention in constructing electrochemical sensors because of their outstanding electrocatalytic performance. Up to now, most of the researches were focused on the electrochemical application of single heteroatom doped carbon microspheres. In this work, N, P-co-doped carbon microspheres (NP-CMs) modified electrode was developed as highly sensitive electrochemical sensor for acetaminophen. NP-CMs were acquired by the calcination of products from the one-pot reaction of dopamine and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO). Experimental results revealed that NP-CMs showed better electrocatalytic performance compared with N-doped carbon microspheres. Under the optimal conditions, the response of NP-CMs/GCE showed a linear relationship with acetaminophen concentration from 0.07 mu M to 30 mu M, the linear equation was Y = 0.01356X+0.7417 and the detection limit was 40 nM (S/N = 3). The recoveries of the NP-CMs modified GCE for the assay of acetaminophen in human serum samples were from 94.0% to 106.0%. It was believed that N, P-co-doped carbon microspheres have a great potential in the design of novel pharmaceutical analysis methods. (C) 2019 The Electrochemical Society.