【Objectives】Information on long-term impacts of partial substitution of urea, maize straw with manure and straw biochar on agro-ecosystem greenhouse gas emissions is lacking under field condition. 【Methods】Partial substitution of urea, maize straw with manure and straw biochar was designed in a high fertile black soil under spring-maize in Heilongjiang Province, China from 2013 to 2015. Four treatments in quadruplicated plots were used: applying 4 t/hm~2 of maize straw in N0, N1 and N2, and 2 t/hm~2 maize straw biochar in N3; applying urea N 165 kg/hm~2 for N1; applying 60% urea N + 20% manure N + 20% slow-released N for N2; The same N input with N2 was applied in N3 except straw biochar replacing maize straw. Greenhouse gas (CO_2, N_2O and CH4) emissions were measured during maize growth season. Maize yield, aboveground biomass and soil organic matter content in surface layer were measured after maize harvest. 【Results】There was no significant difference in maize yield among fertilizer treatments. Under equal N input condition, CO_2 emissions in N1, N2 and N3 were 13170, 10521 and 9994 kg/hm~2, respectively. And N2 and N3 significantly decreased CO_2 emission compared with N1 treatment (P < 0.05). The cumulative N_2O emission in N1, N2 and N3 was 6.092, 6.597 and 3.604 kg/hm~2, respectively. And N3 significantly decreased N_2O emission compared with other treatments. The cumulative methane emissions in N1, N2 and N3 were 0.694, 1.652 and -2.107 kg/hm~2, respectively. And the methane emission under N3 was significantly decreased compared with other treatments. Based on net carbon budget of agro-ecosystem (NECB), N2 (C 766.5 kg/hm~2) was C sink, while N1 (C -621.3 kg/hm~2) and N3 (C -673.3 kg/hm~2) were C sources. One-season soil C sequestration potential estimated with NECB wasC -142.9, 176.3 and 1385.1 kg/hm~2 in N1, N2 and N3, respectively. And the soil C sequestration potential under N3 was significantly larger than those of N2 and N1. Given carbon emission from farming practices and chemical inputs included, chemical N production was the main source, which contributed 73%, 71% and 66% to CO_2 production from total agricultural management practices in N1, N2 and N3, respectively. Based on net global warming potential (Net GWP), which balancing CO_2 emission from agricultural management, chemical inputs, greenhouse gas emissions and soil C sequestration, net CO_2 emissions under N1, N2 and N3 were 2535.2, 1488.2 and -3769.7 CO_2 eq. kg/hm~2, respectively. This means only N3 treatment was C sink based on Net GWP. 【Conclusions】In the black soil, partial substitution of chemical N with manure showed a positive potential for carbon budget in agro-ecosystem. Substitution of maize straw with straw biochar significantly increased soil C sequestration potential and decreased N_2O emission. Therefore, combining manure and straw biochar was an efficient choice for improving soil C sequestration and mitigating greenhouse effect.