Dozens of Buddhist monks and demonstrators waving national flags and signs reading "No Rohingya" congregated at the Thilawa port waiting for the ship to dock.
Hundreds of Rohingya are thought to have been killed in a brutal four-month campaign by security forces that the UN says may amount to ethnic cleansing.
Tens of thousands have fled to neighbouring Bangladesh bringing harrowing tales of murder and rape.
"We want to let them know that we have no Rohingya here," a Buddhist monk named Thuseitta, from the Yangon chapter of the Patriotic Myanmar Monks Union, told AFP at the docks.
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Buddhist nationalist groups are especially strong in their vitriol, portraying them as illegal immigrants from Bangladesh.
Myanmar's treatment of the Rohingya has sparked criticism from Muslim-majority Malaysia, in a rare spat between Southeast Asian neighbours.
The Nautical Aliya set off from Malaysia last week carrying 2,200 tonnes of rice, medical aid and clothing along with hundreds of health workers and activists.
Part of the aid will be unloaded in Yangon and transported overland to the north of Rakhine state, site of the military crackdown.
Myanmar initially refused to allow the ship into its waters and has barred it from sailing to Rakhine's state capital Sittwe.
The government has also demanded that the aid be distributed to both Rohingya and Buddhist ethnic Rakhines.
The delivery comes days after a blistering report from the UN accused Myanmar's security forces of carrying out a campaign of rape, torture and mass killings against the Rohingya.
Based on interviews with hundreds of escapees in Bangladesh, investigators said the military's "calculated policy of terror" very likely amounted to ethnic cleansing.
The UN's top official on preventing genocide, Adama Dieng, said this week that a government commission tasked with investigating allegations of abuse was "not a credible option".