Grégory Villemin was four when his body was found in the Vologne river. His killer has never been found.
All three held by police were described as relatives of the boy's father.
The murder has continued to haunt France ever since, but advances in DNA technology have helped police shed further light on the case.
It was on 16 October 1984 that Grégory Villemin's body was found and a magistrate opened what became one of the biggest mysteries in French criminal history.
Hours before the boy was discovered, his hands and feet tied, his uncle said he had received a phone call from someone claiming to have kidnapped him. The following day, the child's parents received a letter that said: "Your son is dead, I have been avenged."
The next month, a cousin of the boy's father, Bernard Laroche, was arrested when his sister-in-law, Muriel Bolle, testified against him. He was released in 1985 when she retracted her statement, but shot dead by Jean-Marie Villemin, the boy's father, weeks later.
The father went to jail for Laroche's murder for several years. The boy's mother, Christine Villemin, was also jailed for her son's murder but later cleared. Both were later given compensation by the state for miscarriage of justice.
The anonymous phone-calls and poison-pen letters continued over the years, and the case was reopened first in 2000 and then again in 2008 in an attempt to identify the DNA on the letters. Three separate traces of DNA were found but not identified.
Little news has been heard in the "Grégory affair" since police said in 2013 that DNA tests had brought the investigation no further.
That was until about 08:00 (06:00 GMT) on Wednesday, when investigators from Dijon detained a couple in their 70s in the village of Aumontzey, in the Vologne river valley. Sources told French media that they were the father's uncle and aunt.
They were placed in police custody on suspicion of complicity in the murder, failing to report a crime or helping a person at risk, according to L'Est Républicain website.
A third person reported to be the father's half-sister was also being held. A fourth, said to be the victim's grandmother, was taken in as a witness. Her husband was also questioned.
Prosecutor Jean-Jacques Bosc said investigators had targeted "people very close to the heart of this case" and hoped to provide answers to the questions they had.
Local reporters covering the case pointed out that none of those held on Wednesday was suspected of the boy's murder.
They also said that Muriel Bolle, who as a teenager had initially blamed the killing on Bernard Laroche, had been asked to provide a DNA sample on Wednesday before later being released.
A couple in their 70s and a woman have been detained for questioning by police, 32 years after a crime that horrified France.